Control of growth and repair of gastro-intestinal tissues by gastrokines and inhibitors

ABSTRACT

A novel group of gastrokines called Gastric Antrum Mucosal Protein is characterized. A member of the group is designated AMP-18. AMP-18 genomic DNA, cDNA and the AMP-18 protein are sequenced for human, mouse and pig. The AMP-18 protein and active peptides derived from it are cellular growth factors. Surprisingly, peptides capable of inhibiting the effects of the complete protein, are also derived from the AMP-18 protein. Cytoprotection and control of mammalian gastro-intestinal tissue growth and repair (restitution) is facilitated by the use of the proteins, making the proteins candidates for therapies in inflammatory bowel disease, mucositis, and gastric ulcers.

This application is a continuation-in-part of U.S. Ser. No. 10/473,571filed Sep. 29, 2003 and U.S. Ser. No. 09/821,766 which issued May 11,2004 as U.S. Pat. No. 6,734,289, and also claims priority to PCT/US02/09885, filed Mar. 29, 2002.

BACKGROUND

Searches for factors affecting the mammalian gastro-intestinal (GI)tract are motivated by need for diagnostic and therapeutic agents. Aprotein may remain part of the mucin layer, providing mechanical (e.g.,lubricant or gel stabilizer) and chemical (e.g. against stomach acid,perhaps helping to maintain the mucus pH gradient and/or hydrophobicbarrier) protection for the underlying tissues. The trefoil peptidefamily has been suggested to have such general cytoprotectant roles (seeSands and Podolsky, 1996). Alternatively, a cytokine-like activity couldhelp restore damaged epithelia. A suggestion that the trefoil peptidesmay act in concert with other factors to maintain and repair theepithelium, further underlines the complexity of interactions that takeplace in the gastrointestinal tract (Podolsky, 1997). The maintenance ofthe integrity of the GI epithelium is essential to the continuedwell-being of a mammal, and wound closing after damage normally occursvery rapidly (Lacy, 1998), followed by proliferation and differentiationsoon thereafter to reestablish epithelial integrity (Nursat et al.,1992). Thus protection and restitution are two critical features of thehealthy gastrointestinal tract, and may be important in the relativelyharsh extracellular environment of the stomach.

Searches for GI proteins have met with some success. Complementary DNA(cDNA) sequences to messenger RNAs (mRNA) isolated from human andporcine stomach cells were disclosed in the University of Chicago Ph.D.thesis “Characterization of a novel messenger RNA and immunochemicaldetection of its protein from porcine gastric mucosa,” December 1987, byone of the present inventors working with the other inventors. However,there were several cDNA sequencing errors that led to significant aminoacid changes from the AMP-18 protein disclosed therein. The proteinitself was isolated and purified only as an aspect of the presentdisclosure, and functional analyses were performed to determine utility.Nucleic acid coding sequences were sought.

SUMMARY OF THE DISCLOSURE

A novel group of Gastric Antrum Mucosal Proteins that are gastrokines,is characterized. A member of the gastrokine group is designated AMP-18.AMP-18 genomic DNA, and cDNA molecules was sequenced for human andmouse, and the protein sequences are predicted from the nucleotidesequences. The cDNA molecule for pig AMP-18 was sequenced and confirmedby partial sequencing of the natural protein. The AMP-18 protein andactive peptides derived from its sequence are cellular growth factors.Surprisingly, peptides capable of inhibiting the effects of the completeprotein, were also derived from the AMP-18 protein sequence. Control ofmammalian gastro-intestinal tissues growth and repair was facilitated bythe use of the protein or peptides, making the protein and the derivedpeptides candidates for therapies.

The protein was discovered in cells of the stomach antrum mucosa byanalysis of cDNA clones obtained from humans, pigs, and mice. Theprotein is a member of a group of cellular growth factors or cytokines,more specifically gastrokines. The AMP-18 cDNA sequences predict aprotein 185 amino acids in length for both pig and man. The nucleotidesequences also predict a 20-amino acid N-terminal signal sequence forsecreted proteins. The cleavage of this N-terminal peptide from theprecursor (preAMP-18) was confirmed for the pig protein; this cleavageyields a secreted protein 165 amino acids in length and ca. 18,000Daltons (18 kD) in size. Human and mouse genomic DNA sequences were alsoobtained and sequenced. A human genomic DNA was isolated in 4overlapping fragments of sizes 1.6 kb, 3 kb, 3.3 kb and 1.1 kbrespectively. The mouse genomic DNA sequence was isolated in a singleBAC clone.

The gastrokine designated AMP-18 protein was expressed at high levels incells of the gastric antrum. The protein was barely detectable in therest of the stomach or duodenum, and was not found, or was found in lowlevels, in other body tissues tested. AMP-18 is synthesized in lumenalsurface mucosal cells, and is secreted together with mucin granules.

Studies in humans confirm the location and expression of the AMP-18peptide in human gastric mucosa.

Compositions of AMP-18 isolated from mouse and pig antrum tissuestimulate growth of confluent stomach, intestinal, and kidney epithelialcells in culture; human, monkey, dog and rat cells are also shown torespond. This mitogenic (growth stimulating) effect is inhibited byspecific antisera (antibodies) to AMP-18, supporting the conclusion thatAMP-18, or its products, e.g. peptides derived from the protein byisolation of segments of the protein or synthesis, is a growth factor.Indeed, certain synthetic peptides whose amino acid sequences representa central region of the AMP-18 protein also have growth-factor activity.The peptides also speed wound repair in tissue culture assays,indicating a stimulatory effect on cell migration, the process whichmediates restitution of stomach mucosal injury. Thus, the protein andits active peptides are motogens. Unexpectedly, peptides derived fromsub-domains of the parent molecule can inhibit the mitogenic effect ofbioactive synthetic peptides and of the intact, natural protein presentin stomach extracts.

There are 3 activities of the gastrokine proteins and peptides of thepresent invention. The proteins are motogens because they stimulatecells to migrate. They are mitogens because they stimulate celldivision. They function as cytoprotective agents because they maintainthe integrity of the epithelium (as shown by the protection conferred onelectrically resistant epithelial cell layers in tissue culture treatedwith damaging agents such as oxidants or non-steroidal anti-inflammatorydrugs NSAIDs).

The synthesis of AMP-18 is confined to lumenal mucosal lining epithelialcells of the gastric antrum of humans and other mammals. Inside cellsthe protein is co-localized with mucins in secretion granules, andappears to be secreted into the mucus overlying the apical plasmamembrane. Recombinant human AMP-18 in E. coli exerts its mitogeniceffect at a concentration an order of magnitude lower thangrowth-promoting peptides derived from the center of the mature protein.Peptide 77-97 (SEQ ID NO: 12), the most potent of the mitogenicpeptides, appears to be cell-type specific as it does not stimulategrowth of fibroblasts or HeLa cells. Mitogenesis by specific AMPpeptides appears to be mediated by a cell surface receptor becausecertain peptides that are not active mitogens can competitively inhibit,in a concentration-dependent manner, the growth-stimulating effects ofpeptide 58-99 and antrum cell extracts. AMP-18 and its derived peptidesexhibit diverse effects on stomach and intestinal epithelial cells whichsuggest they could play a critical role in repair after gastric mucosalinjury. These include cytoprotection, mitogenesis, restitution, andmaturation of barrier function after oxidant-and/orindomethacin-mediated injury. Possible mechanisms by which AMP-18 or itspeptide derivatives mediate their pleiotropic effects includestimulation of protein tyrosine kinase activity, prolongation of heatshock protein expression after cell stress, and enhanced accumulation ofthe tight junction-associated protein ZO-1 and occludin. Certain ofthese physiological effects can occur at concentrations that arerelatively low for rhAMP-18 (<50 nM) compared to the concentrations ofother gastric peptide mediators such as trefoil peptides or theα-defensin, cryptdin 3 (>100 μM). Immunoreactive AMP-18 is apparentlyreleased by cells of the mouse antrum after indomethacin gavage, and bycanine antrum cells in primary culture exposed to forskolin, suggestthat the protein is subject to regulation. These results imply thatAMP-18 could play a role in physiological and pathological processessuch as wound healing in the gastric mucosal epithelium in vivo.

A group of isolated homologous cellular growth stimulating proteinsdesignated gastrokines, are produced by gastric epithelial cells andinclude the consensus amino acid sequences VKE(K/Q)KXXGKGPGG(P/A)PPK(SEQ ID NO: 10) wherein XX can be LQ or absent (which results in SEQ IDNOS 25 and 26, respectively). An isolated protein of the group has anamino acid sequence as shown in FIG. 7. The protein present in piggastric epithelia in a processed form lacking the 20 amino acids whichconstitute a signal peptide sequence, has 165 amino acids and anestimated molecular weight of approximately 18 kD as measured bypolyacrylamide gel electophoresis. Signal peptides are cleaved afterpassage through endoplasmic reticulum (ER). The protein is capable ofbeing secreted. The amino acid sequence shown in FIG. 3 was deduced froma human cDNA sequence. An embodiment of the protein is shown with anamino acid sequence as in FIG. 6, a sequence predicted from mouse RNAand DNA.

A growth stimulating (bioactive) peptide may be derived from a proteinof the gastrokine group. Bioactive peptides rather than proteins arepreferred for use because they are smaller, consequently the cost ofsynthesizing them is lower than for an entire protein.

In addition, a modified peptide may be produced by the following method:

-   -   (a) eliminating major protease sites in an unmodified peptide        amino acid sequence by amino acid substitution or deletion;        and/or    -   (b) introducing into the modified amino acid analogs of amino        acids in the unmodified peptide.

A synthetic growth stimulating peptide, has a sequence of amino acidsfrom positions 78 to 119 as shown in FIG. 3.

Another peptide has a sequence of amino acids from position 97 toposition 117 as shown in FIG. 3.

Another peptide has a sequence of amino acids from position 97 toposition 121 as shown in FIG. 3.

Another peptide has a sequence of amino acids from position 104 toposition 117 as shown in FIG. 3.

An embodiment of an isolated bioactive peptide has one of the followingsequences: KKLQGKGPGGPPPK (SEQ ID NO: 11), LDALVKEKKLQGKGPGGPPPK (SEQ IDNO: 12), or LDALVKEKKLQGKGPGGPPPKGLMY (SEQ ID NO: 13). An embodiment ofan inhibitor of a protein of the gastrokine group has the amino acidsequence KKTCIVHKMKK (SEQ ID NO: 14) or KKEVMPSIQSLDALVKEKK. (SEQ ID NO:15) (see also Table 1)

A pharmaceutical composition includes at least one growth stimulatingpeptide.

A pharmaceutical composition for the treatment of diseases associatedwith overgrowth of gastric epithelia, includes an inhibitor of at leastone protein of the group of gastrokines or of a growth stimulatingpeptide derived from the gastrokine proteins.

A pharmaceutical composition for the treatment of diseases of the colonand small intestine includes at least one growth stimulating peptide ofthe present invention. Examples of such diseases include ulcerativecolitis and Crohn's Disease.

Antibodies to the protein product AMP-18 encoded by the human cDNAexpressed in bacteria were produced in rabbits; these antibodies reactedwith 18 kD antrum antigens of all mammalian species tested (human, pig,goat, sheep, rat and mouse), providing a useful method to detectgastrokines. An antibody to a protein of the group recognizes an epitopewithin a peptide of the protein that includes an amino acid sequencefrom position 78 to position 119 as in FIG. 3.

An isolated genomic DNA molecule has the nucleotide sequence of a humanas shown in FIG. 1 and an isolated cDNA molecule encoding a humanprotein, has the nucleotide sequence as shown in FIG. 2.

An isolated DNA molecule has the genomic sequence found in DNA derivedfrom a mouse, as shown in FIG. 4.

Genomic DNA has value because it includes regulatory elements forgastric expression of genes, consequently, the regulatory elements canbe isolated and used to express other gene sequences than gastrokines ingastric tissue.

A mouse with a targeted deletion in a nucleotide sequence in the mousegenome that, when expressed without the deletion, encodes a protein ofthe group of gastrokines of the present invention.

A method of making a gastrokine protein or a peptide derived from agastrokine protein includes:

-   -   a) obtaining an isolated cDNA molecule with a sequence such as        that shown in FIG. 2;    -   (b) placing the molecule in a recombinant DNA expression vector;    -   (c) transfecting a host cell with the recombinant DNA expression        vector;    -   (d) providing enviromnental conditions allowing the transfected        host cell to produce a protein encoded by the cDNA molecule; and    -   (e) purifying the protein from the host cell.

Host cells in which expression has been successful include baculovirus,which allows large amounts of gastrokines to be provided for commercialand research uses. For example, human AMP-18 protein without the signalpeptide was produced.

A recombinant human protein AMP-18 expressed in E. coli has the sequencein FIG. 14, left panel.

A method to stimulate growth of epithelial cells in the gastrointestinaltract of mammals includes:

-   -   (a) contacting the epithelial cells with a composition        comprising a gastrokine protein or a peptide derived from a        protein of the group; and    -   (b) providing environmental conditions for stimulating growth of        the epithelial cells.

A method to inhibit cellular growth stimulating activity of a protein ofthe group includes:

-   -   (a) contacting the protein with an inhibitor; and    -   (b) providing environmental conditions suitable for cellular        growth stimulating activity of the protein.

The inhibitor may be an antibody directed toward at least one epitope ofthe protein, e.g. an epitope with an amino acid sequence from position78 to position 119 of the deduced amino acid sequence in FIG. 3 or aninhibitor peptide such as those in Table 1.

A method of testing the effects of different levels of expression of aprotein on mammalian gastrointestinal tract epithelia, includes thesteps of:

-   -   (a) obtaining a mouse with an inactive or absent gastrokine        protein;    -   (b) determining the effects of a lack of the protein in the        mouse;    -   (c) administering increasing levels of the protein to the mouse;        and    -   (d) correlating changes in the gastrointestinal tract epithelia        with the levels of the protein in the epithelia.

Kits are contemplated that will use antibodies to gastrokines to measuretheir levels by quantitative immunology. Levels may be correlated withdisease states and treatment effects.

A method to stimulate migration of epithelial cells after injury to thegastrointestinal tract of mammals, includes:

-   -   (a) contacting the epithelial cells with a composition        comprising a peptide derived from the protein; and    -   (b) providing environmental conditions allowing migration of the        epithelial cells.

A method for cytoprotection of damaged epithelial cells in thegastrointestinal tract of mammals, includes:

-   -   (a) contacting the damaged epithelial cells with a composition        including a protein of the gastrokine group or a peptide derived        from the protein; and    -   (b) providing environmental conditions allowing repair of the        epithelial cells.

The damaged cells may form an ulcer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a human genomic nucleotide sequence (SEQ ID NO: 1) of apre-gastrokine; sequence features were determined from cDNA and PCR ofhuman genomic DNA amph-ge8.seq. Length: 7995 predicted promoter: 1405;exon 1: 1436-1490; exon 2: 4292-4345; exon 3: 4434-4571; exon 4:5668-5778; exon 5: 6709-6856; exon 6: 7525-7770; polyA site: 7751 (amphrefers to antrum mucasal protein human genomic nucleotide sequence.

FIG. 2 is a human cDNA sequence (SEQ ID NO: 2); the DNA clone wasobtained by differential expression cloning from human gastric cDNAlibraries.

FIG. 3 is a human preAMP-18 protein sequence (SEQ ID NO: 3) predictedfrom a cDNA clone based on Powell (1987) and revised by the presentinventors; N-21 is the expected N-terminus of the mature protein.

FIG. 4 is a mouse preAMP-18 sequence (SEQ ID NO: 4) determined fromRT-PCR of mRNA and PCR of BAC-clones of mouse genomic DNA sequences:

-   -   predicted promoter: 1874 experimental transcription start site:        1906 translation initiation site: 1945 CDS 1: 1906-1956; CDS 2:        3532-3582; CDS 3: 3673-3813; CDS 4: 4595-4705; CDS 5: 5608-5749;        CDS 6: 6445-6542; polyA site: 6636.

FIG. 5 is a mouse cDNA sequence (SEQ ID NO: 5) for preAMP-18.

FIG. 6 is mouse preAMP-18 amino acid sequence (SEQ ID NO: 6); RT-PCRperformed on RNA isolated from mouse stomach antrum: Y-21 is thepredicted N-terminus of the mature protein; the spaces indicated by . .. mean there are no nucleotides there to align with other sequences inFIG. 11.

FIG. 7 is a cDNA expressing porcine AMP-18 (SEQ ID NO: 7).

FIG. 8 is pig pre-gastrokine (pre-AMP-18) protein sequence (SEQ ID NO:8) predicted from a cDNA clone based on Powell (1987) D-21 is theN-terminus of the mature protein—confirmed by sequencing of the proteinisolated from pig stomach.

FIG. 9 is a comparison between the amino acid sequences of human (SEQ IDNO: 3) versus pig (SEQ ID NO: 8) pre-gastrokine.

FIG. 10 shows a computer-generated alignment comparison of human (SEQ IDNO: 3), pig (SEQ ID NO: 8) and mouse (SEQ ID NO: 6) predicted proteinsequences determined from sequencing of cDNA clones for human and pigAMP-18, and by polymerase chain reaction of mouse RNA and DNA usingpreAMP-18 specific oligonucleotide primers; in each case the first 20amino acids constitute the signal peptide, cleaved after passage throughthe endoplasmic reticulum membrane.

FIG. 11 shows the effect of porcine gastric antrum mucosal extract,human AMP peptide 77-97, of the mature protein (same as peptide 97-117of the human precursor protein: Table 1) and EGF on growth of gastricepithelial cells; AGS cells were grown in DMEM containing fetal bovineserum (5%) in 60-mm dishes; different amounts of pig antrum extract,HPLC purified peptide 77-97, and/or EGF were added; four days later thecells were dispersed and counted with a hemocytometer; antrum extractand peptides each stimulated cell growth in a concentration-dependentmanner; the bar graph shows that at saturating doses, peptide 77-97 (8μg/ml) or EGF (50 ng/ml) was mitogenic; together they were additivesuggesting that the two mitogens act using different receptors and/orsignaling pathways; anti-AMP antibodies inhibited the antrum extract butdid not inhibit peptide 77-97.

FIG. 12 shows the structure of the human and mouse preAMP-18 genes; thenumber of base pairs in introns are shown above the bars; exons areindicated E1-E6 and introns I1-I5; there are minor differences in intronlength.

FIG. 13 shows Left panel. Amino acid sequence of recombinant humanAMP-18 (residues 21-185 of SEQ ID NO: 3) expressed in E. coli. Note theHis6-tag (SEQ ID NO: 16) within a 12 amino acid domain (SEQ ID NO: 9) atthe N-terminus that has replaced the putative hydrophobic signalpeptide. RightpaneL Effect of rhAMP-18 and AMP peptide 77-97 on growthof confluent cultures of IEC-18 cells. Although maximal growthstimulation is similar, the half-maximal concentration (K_(1/2)) forrhAMP-18 (˜30 nM) is about an order of magnitude lower than for thepeptide (˜300 nM).

FIG. 14 shows Left Panel. Alignment of the open reading frames (ORF)derived from the cDNA clones for AMP-18 for the precursor proteins ofhuman (SEQ ID NO: 3) and pig (SEQ ID NO: 8) antrum. Similarity was78.50% and identity was 75.27%. Computer analysis was carried out usingthe GAP and PEPTIDESTRUCTRE programs of the Wisconsin Package (GCG).Right Panel Model of the predicted secondary structure for the humanpreAMP ORF. Attention is drawn to the asparagine rich N-terminal domain,the short tryptohopan (W)-rich and glycine-proline (GP) regions, and theconserved positions of the four cysteine (C) residues. Possibleamphipathic helices are indicated.

FIG. 15 shows the effect of porcine antrum cell extract, peptide 77-97,and EGF on growth of intestinal epithelial cells. IEC-6 cells were grownin 60-mm dishes. Antrum cell extract (left panel) and peptide 77-97(centerpanel) each stimulated growth in a concentration-dependentmanner. Peptide 77-97 (1 μg/ml) appeared more potent than EGF (50 ng/ml)(right panel). Values are means±SE for 3 cultures.

FIG. 16 shows the effect of AMP peptide 77-99 and EGF on growth andwound restitution by human antrum epithelial cells. To measure growth(left panel), HAE cells were plated in 60-mm dishes. Peptide 77-97 (8μg/ml), or EGF (50 ng/ml), or both were added to the medium and thenumber of cells counted 4 days later. Peptide 77-97 and EGF eachstimulated proliferation, and appeared to be additive. Values aremeans±SE for 3 cultures. To measure migration (right panel), cells weregrown in 60-mm dishes to prepare a confluent monolayer. The medium wasaspirated and replaced with fresh medium containing 0.01% calf serum(CS). The monolayer was mechanically wounded by scraping with a razorblade. Detached cells were removed by aspirating medium, and rinsing theremaining cells twice with fresh medium containing 0.01% CS. Freshmedium (5 ml) containing CS (0.01%) and insulin (100 U/L) was added towounded cultures. Either peptide 77-97 (8 μg/ml), EGF (50 ng/ml), orboth were added to duplicate cultures. Migration was assessed at 24, 48and 72 hr after wounding by measuring the distance (in mm) that cellshad migrated from the wound edge using a microscope eyepiece reticle(10-mm long; 0.1-mm markings). Migrating cells at 12 randomly chosensites along a 0.25-mm stretch of the wound edge were measured at 40-foldmagnification. Migration at 2 different sites was measured for each of 2separate wounds made in each culture. Values are the mean distance cellsmoved into the denuded area from the edge of 4 different wounds in 2cultures±SE. Cells exposed to peptide 77-97 migrated further from thewound edge than those exposed to vehicle at 72 hr. EGF also stimulatedcell movement, and the two agents acting together markedly enhancedmigration.

FIG. 17 shows the effect of AMP peptide 67-85 on growth of intestinalepithelial cells stimulated by peptide 58-99. Confluent cultures ofIEC-18 cells were prepared. One day later, medium was aspirated andreplaced with 5 ml of DMEM containing CS (0.5%) and insulin, without(control) or with mitogenic peptide 58-99 (8 μg/ml). Sister platesreceiving 1 ml medium and different amounts of peptide 67-85 wereincubated at 1 hr at 38° C. on a CO₂ incubator, and then an additional 4ml of medium was added to each dish. Peptide 58-99 was added to 2 of the4-sister plates at each concentration of peptide 67-85, and the numberof cells was counted. In the absence of peptide 67-85, cell numberincreased by 290%, whereas cells exposed to peptide 58-99 increased innumber by 407%, and EGF-treated (50 ng/ml) cells increased by 402%during the next 3 days. Stimulation of cell growth by mitogenic peptide58-99 was completely abolished by preincubation of cells with 0.25 μg/mlof peptide 67-85. When added alone, peptide 67-85 (0.25 to 8 μg/ml) wasnot a mitogen. Values for the number of cells per culture are shownrelative to multiplication of cells exposed to the vehicle during thesame period.

FIG. 18 shows the effect of rabbit antiserum to AMP-18 on mitogeniceffect of rhAMP-18 on confluent IEC-18 cells. When rhAMP-18 (50nanomolar) was preincubated for 30 min with antiserum (1:100dilution)+Ab), growth stimulation was reduced by ˜95%; preimmune serumhad no effect on cell growth. The half-maximal concentration (K_(1/2))for growth stimulation of this recently purified rhAMP-18 is about 5nanomolar.

FIG. 19 shows the effect of AMP peptide 77-97 on wound restitution inhuman antrum (HAE) and rat intestinal (IEC-18) epithelial cells.Confluent monolayer cultures were mechanically wounded by scraping witha razor blade, and the distance that cells migrated from the wound edgewas measured using a microscope eyepiece reticle. Cells migrated furtherin the presence of AMP peptide at each time point studied (P<0.005).

FIG. 20 shows the effect of AMP peptide 77-97 on maturation of TER.Monolayer cultures of MDCK cells were grown on permeable polycarbonatefilters (0.4-μm pore size) (Transell) in DMEM containing FBS (2%)without (control) or with peptide 77-97 (8 μg/ml) for 8 days. TER wasmeasured 24 hr after the cells were plated, and at specified timesthereafter using an epithelial volt-ohm meter (EVOM, Millipore).Following each measurement, medium containing FBS without or withpeptide was changed (0, 48, and 144 hr), and additional peptide 77-97 (8μg/ml) was added at 30 and 72 hr. At 72 hr, TER in cultures thatreceived peptide 77-97 was twice as high as in control cultures. Valuesare means for 3 cultures; variance is <10% of the mean. TER was measuredfrom 3 different areas on the filter.

FIG. 21 shows the effect of AMP peptide 77-97 on TER in monolayersinjured with the oxidant monochloramine or indomethacin. Panel A: When astable TER was reached (330 Ω·cm²) in MDCK cell monolayers the mediumwas changed to DMEM containing FBS (0.2%), and either peptide 77-97 (8μg/ml) or EGF (50 ng/ml). After 18 hr, peptide 77-97 or EGF were addedto the specified wells. One hour later monochloramine (0.1 mM), like theother agents, was added to the apical and basal compartments of theTranswell. Monochlorarnine-injured cultures treated with vehicle or EGFsustained ˜35-40% loss of TER 90 min after oxidant exposure, whereas theTER of oxidant-injured cells treated with peptide 77-97 was similar tocontrol cultures not exposed to the oxidant. Panels B, C: Caco2/bbe (C2)subclone monolayers were grown on collagen-coated polycarbonate filtersuntil a stable TER was reached (225 Ω·cm²). Spent medium was replacedwith fresh medium containing FBS (0.1%) alone or with peptide 77-97 (8μg/ml). After 18 hr, monochloramine (0.3 mM, B) or indomethacin (0.1 mM,C) was added to both compartments of the Transwell. At time 0, culturesreceived either vehicle (control), vehicle plus oxidant or indomethacin,or peptide 77-97 and oxidant or indomethacin. TER of injured culturestreated with vehicle decreased by ˜35% at 90 min, whereaspeptide-treated cultures declined ˜10%. The peptide did not alter TER ofnon-injured cells.

FIG. 22 shows the effect of AMP peptide 77-97 on TER following injury byDSS. C2 cell monolayers were grown in DMEM containing FBS (5%) andtransferrin (10 μg/ml) on collagen-coated polycarbonate filters until astable TER was reached (225 Ωcm²). At time 0, cells were exposed to noDSS (control), or DSS (4%) in the upper compartment of the Transwell.AMP peptide 77-97 (8 μg/ml) was added to the upper and lowercompartments of the Transwell 1 day prior to the addition of DSS at time0. TER of DSS-injured cultures treated with vehicle decreased by ˜70% at45 min, whereas peptide-treated cultures declined ˜10% at that time. Thepeptide did not alter TER of non-injured cells. Values are means for ≧6cultures.

FIG. 23 shows the effect of AMP peptide 77-97 on ZO-1 and occludin afteroxidant injury of C2 cells. This immunoblot shows that protein levels inthe insoluble fraction are˜two-fold greater after exposure of cells toAMP peptide than to the vehicle.

FIG. 24 shows the effect of AMP peptide on C2 cells. Cultures wereexposed to the peptide for different periods of time and the insolublefraction was obtained. Proteins were separated, immunoblots were probedwith specific antisera, and the amount of each protein was quantifiedusing laser densitometry.

FIG. 25 shows the effect of rhAMP-18 on TER of monolayers subjected tooxidant injury. Confluent C2 cell monolayers were prepared on Transwellsuntil a stable TER was established. Medium was replaced with freshmedium containing FBS (0.1%) alone (control), or with either rhAMP-18(100 nanomolar) or peptide 77-97 (3.7 micromolar). After 18 hr,monochloramine (0.3 mM) was added to both compartments of the Transwell,and cultures received either vehicle (control), vehicle plus oxidant,rhAMP-18 and oxidant, or peptide 77-97 and oxidant, after which TER wasmeasured.

FIG. 26 shows the effect of rhAMP-19 on levels of ZO-1 and occluding inC2 cells. Monolayer cultures were treated with rhAMP-19 (100 nanomolar)or the vehicle for 8 hr. Following cell lysis, an insoluble(particulate) fraction representing cell membranes andcytoskeleton-associated TJ protein was prepared and then subjected toimmunoblotting. The amount of immunoreactive ZO-1 and occludin is abouttwo-fold greater in rhAMP-18-treated (+) cells than vehicle-treated (1)cells as estimated by laser densitometry of the same immunoblot. Equalprotein loading in each lane was documented by re-probing the blot withan antibody to heat shock protein 73 which is constitutively expressedby these cells.

FIG. 27, Left Panel: Effect of AMP peptide on appearance of blood in thestool of mice with DSS-induced colitis. Mice (n=50) were given 3%dextran sulfate sodium, and stools were assayed daily for the presenceof blood. Appearance of bloody diarrhea was delayed in animals treatedwith AMP peptide (10 mg/kg body weight/day) compared to those given thevehicle (P<0.01). Right panel: Effect of treatment with AMP peptide onbody weight of mice with DSS colitis. After animals (n=20) received 3%DSS to drink for 4 days, they were switched to water (day 0 on graph).Mice given AMP peptide daily (10 mg/kg, s.c.) lost less weight thanthose given vehicle during the next 3 days (P<0.01). AMP peptide-treatedmice completely recovered from the adverse effect of DSS by day 7,whereas animals given the vehicle did not (P<0.01).

FIG. 28 shows that AMP peptide 77-97 inhibits growth of humanHelicobacter pylori. When a lawn of H. pylori was prepared on a culturedish, growth of the organisms was inhibited by the antibioticclarithromycin (positive control) and at 3 concentrations of AMP peptide77-97, but not by scrambled AMP peptide.

FIG. 29. AMP peptide speeds recovery of TER and restitution afterDSS-mediated injury in cultures of C2 cells. After DSS reduced TER (leftpanel) or cell migration in scrape-wounded cultures (right panel), itwas removed by aspirating the culture medium. Then fresh mediumcontaining AMP peptide (8 μg/ml) or vehicle was added. Recovery of TERand restitution proceeded to a greater extent in the presence of AMPpeptide (P<0.001).

FIG. 30. Effect of AMP peptide on occludin localization by confocalmicroscopy in C2 cells under control conditions and following oxidantinjury. Occludin immunoreactivity in a control cell monolayer (panel A)formed a uniform band outlining the cell junctions that was more intensethan in the cytoplasm. When cells were exposed to AMP peptide for 18 hr(panel B), occludin appeared to be relatively more abundant in the TJsand less in the cytoplasm than in control cells. Following exposure tothe oxidant monochloramine (0.3 mM) for 30 min (panel C), occludinintensity at the cell junctions was reduced and at some sites wasdiscontinuous; occasionally it was barely visible (arrows). In cellsthat were pretreated with AMP peptide prior to the oxidant (panel D),occludin immunoreactivity at the cell junctions was more intense than inuntreated, injured cells. Occludin was visualized with a mouse primaryantibody (1:100 dilution), and Cy3-conjugated AffiniPure goat anti-mouseIgG (1:1000 dilution). Localization of occludin was analyzed using aFluoview 200 laser scanning confocal microscope equipped with a HeNe 533nm laser at 60× magnification. Images were compiled from a Z-series.

FIG. 31. Specificity of AMP peptide stimulation of occludin and ZO-1accumulation in C2 cell monolayers. Confluent cultures were exposed tovehicle, EGF (50 ng/ml), scrambled AMP peptide (8 μg/ml), or intact AMPpeptide (8 μg/ml) for 8 hr. The NP-40 insoluble fraction was prepared,and the amount of occludin and ZO-1 assessed by immunoblotting on a 10%SDS-polyacrylamide gel. Only AMP peptide enhanced accumulation ofoccludin and ZO-1.

FIG. 32. AMP peptide stimulates accumulation of the tightjunction-associated proteins occludin, ZO-1, ZO-2 and claudin-5, and theadherens junction protein E-cadherinin C2 cells after 8 hr.

FIG. 33. Exposure to AMP peptide increases accumulation of junctionaladhesion molecule (JAM) at 48 and 72 hr in C2 cells.

FIG. 34. Cell surface specificity of AMP peptide-stimulated accumulationof tight junction proteins. Exposure of the basolateral but not theapical plasma membrane to AMP peptide is associated with increasedaccumulation of occludin and claudin-5, but not hsc73. This suggeststhat receptors for AMP peptide reside primarily on the basolateralrather than the apical surface of these colonic epithelial (C2) cells.

FIG. 35. A five-fold increase in occludin and a doubling of TER from 132to 278 Ω≅cm² occurs between 1 and 15 days after plating C2 cells. Twoforms of occludin (62 kDa and 74 kDa) were resolved using a 12.5%polyacrylamide gel.

FIG. 36. AMP peptide prevents loss of occludin in DSS-mediated injury ofC2 cells. Occludin immunoreactivity declined by 50% 1 hr after exposureof the cell monolayer to 4% DSS (top right panel) compared tovehicle-treated cells (top left). Treatment with AMP peptide (8 μg/ml)for 18 hr appeared to double the amount of occludin (bottom left) whichwas not reduced in peptide-treated cells exposed to DSS (bottom right).

FIG. 37. AMP peptide protects barrier function following disruption ofthe actin filament network in C2 cells. Cytochalasin D, which disruptsactin filaments, reduced TER by ˜35% in vehicle-treated cells after 2hr, whereas pretreatment with AMP peptide prevented the decline.

FIG. 38. Administration of 3% DSS to mice for 4 days reducedimmunoreactive occludin by 50% in the colonic mucosa compared to animalsgiven water. The colonic epithelium from each mouse was scraped into atube, and the NP-40 insoluble fraction was obtained and analyzed foroccludin content by immunoblotting. Each bar is the mean±SE for 5 mice(P<0.01).

FIG. 39. Effect of AMP peptide on occludin in mouse colon. AMP peptidetreatment increases accumulation of both high-and low-molecular weightimmunoreactive occludin, but does not alter amount of β-catenin incolonic mucosal epithelial cells.

FIG. 40 shows that treatment of C2 cell monolayers with AMP peptideprevents the fall in TER caused by a lectin (PA-I) derived from thesurface of Pseudomonas aeruginosa. Cells were prepared and treated withAMP peptide as described in the legend to FIG. 22. The TER of cellsexposed to PA-I and treated with the vehicle declined by ˜22% at 4 hr,whereas the TER of peptide-treated cultures was nearly unchanged at thattime.

FIG. 41 shows that AMP peptide prevents death in mice with Pseudomonasaeruginosa-induced gut-derived sepsis. Balb/c mice were subjected tostress (partial hepatectomy and food deprivation after surgery), andreceived an injection of living P. aeruginosa into the cecum. One daylater all of the mice given AMP peptide were alive, whereas all animalsgiven the vehicle died.

FIG. 42. Effect of AMP peptide on expression of heat shock proteins byintestinal epithelial cells after thermal injury. IEC-18 cell monolayerswere subjected to non-lethal heat shock injury by exposure to 42° C. for23 min. Immediately thereafter some cultures received AMP peptide (8μg/ml) (P on figure); control cultures (C) received vehicle. Whole cellprotein was analyzed by immunoblotting. Increased expression of hsp25and induced hsp72 is seen 12 hr after heat shock in control cultures,but constitutive expression of hsc73 is not altered. Expression of hsp25and 72 was increased after treatment with AMP peptide compared tovehicle at 12 and 24 hr.

FIG. 43. Effect of AMP peptide on tyrosine phosphorylation of IEC-18cell proteins. Cell lysates were prepared, fractionated, blotted, andprobed with anti-phosphotyrosine antibody 4G10. The image isrepresentative of immunoblots from 3 experiments prepared by exposingcells to AMP peptide (8 μg/ml) for 2 min. Protein size markers in kDaare shown on both sides of the figure. Equal protein loading in eachlane was documented by probing a sister blot with an antibody (1B5) tohsc73 which is constitutively expressed by these cells.

FIG. 44. AMP peptide activates p38 MAP kinase and inducesphosphorylation of hsp25. AMP peptide increased p38 MAP kinasephosphorylation at 30 min with no change in total p38 MAP kinase inEC-18 cells (left panel). Treatment with AMP peptide stimulated theappearance of phospho-hsp25 at 30 min and also increased the amount oftotal hsp25 after 1 hr (right panel).

FIG. 45. JNK1/2 and ERK1/2 are rapidly activated by AMP peptide asindicated by the appearance of phosphorylated JNKs after 10 min (leftpanel) and ERKs at 20 min (right panel).

FIG. 46. Steps in AMP peptide-mediated cytoprotection of intestinalepithelial cells. When AMP peptide binds to its putative cell surfacereceptor it stimulates tyrosine phosphorylation of multiple proteins.This is followed by phosphorylation of 3 classes of signaling molecules(p38 MAPK, JNK1/2, ERK1/2), increased accumulation of TJ (occludin,ZO-1, ZO-2, claudin-5), AJ (E-cadherin), and heat shock (hsp25, hsp72)proteins, and functional stabilization of actin. These cellularresponses may contribute to AMP peptide's cytoprotective effect when theintestinal epithelium is subjected to barrier-disrupting agents.

DETAILED DESCRIPTION OF THE DISCLOSURE

Summary

The results disclosed herein characterize the structure and function ofAMP-18 using both a recombinant human protein prepared in E. coli and asynthetic peptide that are both bioactive. The pleiotropic effects ofAMP peptide 77-97 in epithelial cell cultures include maturation,protection and repair of barrier function, as well as stimulation ofrestitution and cell proliferation, all of which relate to protectiveand reparative roles in GI mucosal injury. The cytoprotective effect ofAMP peptide appears to be mediated, at least in part, by its capacity toincrease accumulation of TJ occludin, and other tight and adherensjunction proteins (ZO-1, ZO-2, claudin-5, E-cadherin, JAM), as well ashsp25 and hsp72, and to stabilize the perijunctional actin filamentnetwork after injury (FIG. 46). AMP peptide apparently exerts itseffects via a receptor-mediated mechanism to activate protein tyrosinephosphorylation, and stimulate phosphorylation of p38 MAPK, hsp25, PKCζ,ERK, and JNK. When given to mice, AMP peptide delayed the onset ofbloody diarrhea and protected against weight loss in DSS colitis, andprevented death in gut-derived sepsis. These observations show that thecytoprotective effect of AMP peptide on colonic epithelial cells inculture and in vivo is mediated by the capacity of the peptide toenhance accumulation of specific tight and adherens junction proteinsand hsps, and stabilize actin which could thereby protect and defend thestructure and function of the mucosal barrier in IBD, mucositis,gut-derived sepsis, gastritis, gastric ulcer disease, and theconsequences of gastric antrum infection by H. pylori.

Therapeutic Efficacy

The data disclosed herein points to multiple therapeutic targets forAMP-18/AMP peptide. These include treatment of IBD by: (a) preventing ordecreasing the frequency and intensity of acute exacerbations of thisepisodic disease by the AMP peptide's cytoprotective effect, and (b)speeding recovery of the colonic mucosal epithelium after an attack ofdisease occurs, i.e., a benefit inferred from the mitogenic andmotogenic (wound healing) effects observed in cell culture and murinemodels of colitis. The cytoprotective, mitogenic and motogenic effectsof AMP peptide also predict a therapeutic role in cancer-therapy inducedmucositis of the GI tract as often occurs during chemotherapy and/orradiation therapy. Mucositis occurs in this setting because thetherapeutic protocol is designed to destroy proliferating cancer cells,but may also damage rapidly growing cells that line the mouth, throat,or GI mucosa at any point along its entire length. Injury and/ordestruction of the protective mucosal epithelium can result inlife-threatening infection which puts the patient at risk forgut-derived sepsis and death. Evidence is also provided supporttherapeutic benefits of AMP peptide in the treatment of gut-derivedsepsis (cytoprotection), gastritis and gastric ulcers (cytoprotection,mitogenesis, restitution), and infection with H. pylori (growthinhibition of the organism). The mitogenic and cytoprotective effects ofAMP peptide on renal epithelial cells (MDCK line) in culture disclosedherein also predict therapeutic role for the peptide in patients withacute renal failure.

In summary the cytoprotective, mitogenic, and motogenic effects of AMPpeptide and rhAMP-18 offer multiple therapeutic strategies to preventand/or limit disruption of epithelial barrier function and structure,and also speed regeneration after mucosal injury in gut and kidney.

Other aspects of the disclosure follow.

1. General

A novel gene product, a member of a group of gastrokines, was detectedin mammalian gastric antrum mucosal by a differential screen of cDNAlibraries obtained from different regions of the pig stomach. The cDNAsequence predicted a protein of 185 amino acids including a signalpeptide leader sequence. A cDNA was also isolated from a human library.The predicted amino acid sequence identity between pig and human in76.3%. The sequences predicted a 20 amino acid signal peptidecharacteristic for secreted proteins. The cleavage of this N-terminalsignal peptide was confirmed for the pig protein. Antibodies to theproduct of the human cDNA expressed in bacteria were raised in rabbits;these antibodies reacted with 18-20 OkD antrum antigens of all mammalianspecies tested (pig, goat, sheep, rat and mouse). In agreement with mRNAlevels, the AMP-18 protein is expressed at high levels only in thegastric antrum; it is barely detectable in the rest of the stomach orduodenum, and was not detected in a variety of other tissues tested.AMP-18 is synthesized in the lumenal surface mucosal cells;immuno-electron microscopy locates AMP-18 in the secretion granules ofthese cells. Partially purified AMP-18 preparations from mouse and pigantrum tissue are mitogenic to confluent stomach and kidney epithelialcells in culture; this effect is inhibited by the specific antisera,implying that AMP-18, or its products, is a growth factor.

AMP-18 is likely secreted with the mucus and functions, perhaps aspeptide derivatives within the mucus gel to maintain epithelialintegrity directly, and possibly to act against pathogens. In view ofthe growth factor activity observed on epithelial cell lines in culture,it is likely that AMP-18 or its peptide derivative(s) serves as anautocrine (and possible paracrine) factor for the gastric epithelium.The function of AMP-18 may not be simply as a mitogen, but in additionit may act as differentiation factor providing the signals forreplenishment of the mature lumenal surface cells. The AMP-18 protein orits derivatives are likely important to the normal maintenance of thehighly dynamic gastric mucosa, as well as playing a critical role in therestitution of the antrum epithelium following damage. Limitations ofEST data cannot yield information on starting sequences, signalpeptides, or sequences in the protein responsible for bioactivity, asdisclosed in the present invention. A number of these ESTs have beenreported for mammalian stomach cDNAs, but related ESTs have also beenreported or pancreas and also pregnant uterus libraries. Althoughexpression of AMP-18 RNA in these other tissues appears to be low (asindicated for pancreas by PCR analysis), these results suggest that thisgrowth factor may have broader developmental and physiological rolesthan that implied by the specific high levels of expression found forthe stomach.

The AMP-18 protein appears to be expressed at the surface of thecellular layers of the gastrointestinal (GI) tract. The expressing cellsmay be releasing stored growth factor where needed—in the crypts andcrevices of the GI tract where cellular repair is needed due to surfacedamage.

AMP-18 may act on the mucosal, apical surfaces of the epithelial cells,collaborating with prostaglandins and other growth factors that operatevia basolateral cell surface receptors on the serosal side. The proteinor its derivatives are likely important for the normal maintenance ofthe highly dynamic gastric mucosa, in face of the mechanical stress andhigh acidity of the stomach. AMP-18 may play a critical role in therepair of the stomach epithelium following damage by agents such asalcohol, nonsteroidal anti-inflammatory drugs (NSAIDs), or pathogens, inparticular Heliobacter pylori, which predominantly infects the antrumand is a causative agent of gastric ulcers and possibly cancers.

2. Bioactivity

A synthetic peptide (42 amino acids, a “42-mer”) representing a centralregion of the AMP-18 amino acid sequence also has growth factoractivity, which is inhibited by specific antisera; some related shorterpeptides also have stimulatory activity, while others can inhibit theactivity of the 42-mer. These findings suggest that a saturatableepithelial receptor exists for AMP-18, and opens direct avenues toanalyzing the bioactive regions of the protein and identifying theputative receptor(s). Because AMP-18 does not resemble in structure anyknown cytokine or cytoprotectant protein (such as the trefoil peptides),the analysis of the interactions of the protein, and its active andinhibitory related peptides, with cells offers the opportunity to revealnovel molecular interactions involved in cell growth control.

BSC-1 cell growth was stimulated by gel-fractionated porcine antrumextract; porcine extract protein (250 μg) was loaded into each of 2lanes and subjected to electrophoresis in a polyacrylamide gel (12.5%);the 5 thin slices (2-3 mm) from each area between M_(r) 14 kDa and 21.5kDa were cut from the experimental lanes. Each pair of slices was placedin a silanized microfuge tube with 200 μl sterile PBS, 3% acetonitruleand 1% BSA, and macerated; proteins were eluted from the gel for 18 hrat 22° C. with vigorous shaking; the samples were then microcentrifugedand a sample of a supernatant was added to a confluent culture of BSC-1cells; the number of cells was counted 4 days later; maximal growthstimulation was observed in cultures receiving extracts eluted from gelslices corresponding to a M_(r) of ˜18 kDa; antisera to recombinanthuman AMP-18 added to the culture medium completely inhibited growthstimulation by the 18 kDa fraction (+Ab); values are means of 2cultures; SE is less than 10% of the mean.

The biological activity (mitogenic for epithelial cells in thegastro-intestinal tract) of the AMP-18 is located in the C-terminal halfof the protein. The epitopic sequence(s) appear(s) to be immediatelyN-terminal to the mitogenic sequence.

The biological activity that is a growth factor, is exhibited by apeptide that includes at least 42 amino acids from positions 78 to 119of the full-length protein sequence. An antibody to this region blockedmitogenic activity. Although a peptide having an amino acid sequence of104 to 117 had mitogenic activity, an antibody to this region did notblock (inhibit) the activity. A peptide with an amino acid sequence frompositions 97-117 has the same mitogenic activity as a peptide with the42 amino acid sequence, but is less expensive to produce as a syntheticpeptide.

3. Inhibition of Bioactivity

Epithelial cell growth that was stimulated by murine or porcine antrumcell extract was blocked by rabbit antiserum to a complete, recombinanthuman AMP-18 precursor protein; confluent cultures of BSC-1 cells wereprepared; murine or porcine antrum cell extract was prepared and itsprotein concentration was measured; cell extracts alone and withdifferent dilutions of the antiserum, or antiserum alone (1:100 dilutionwas added to the culture medium, and the number of cells was counted 4days later). Growth stimulation by murine antrum gastrokines wasmaximally inhibited by the antiserum (93%) at a dilution of 1:400,whereas stimulation by the porcine antrum protein extract was totallyinhibited at a dilution of 1:100. Scored values were means for 3cultures; standard error of the mean (SE) was less than 10% of the mean.

Antibodies to the AMP-18 protein have diagnostic uses to determinedifferent levels of the protein in the gastro-intestinal tract in vivo.Ulcers are likely to develop if less than normal levels of AMP-18protein are present. Normal values are determined by technologies knownto those of skill in the art, that is, obtaining representative samplesof persons to be tested (age, sex, clinical condition categories) andapplying standard techniques of protein quantitation. The effects ofaspirin and indamethacin on AMP-18 levels are also useful to monitordeleterious levels of the drugs including the non-steroidalanti-inflammatory drugs (NSAIDs). Stomach cancer cell lines do notexpress the AMP-18 proteins at least by detection methods disclosedherein.

4. Genomic DNA

Genomic AMP-18 DNA sequences have been cloned for human and mouse as aprelude to the analysis of the gene regulatory elements, whichpresumably determine the great differences in the levels of expressionof the gene in tissues where the gene may be active. Upstream anddownstream flanking sequences have been isolated from mouse genomic DNApreparatory to a gene knockout. The flanking genomic sequences likelydetermine the very different levels of expression of the gene in thestomach and few other tissues where it may be expressed. With theinvolvement of different regulatory elements, gastrokine genes could beexpressed as a growth factor in other tissues.

5. Uses of Gastrokines of the Present Disclosure

Because the AMP-18 protein and certain peptides derived from it canstimulate growth and wound repair by stomach and intestinal epithelialcells (as well as kidney) these gastrokine molecules are candidates fortherapeutic agents to speed recovery of the injured GI tract followingpharmacological interventions, radiotherapy, or surgery. In addition,the antibodies developed to gastrokines may be used in kits to measurethe levels of AMP-18 protein or peptide in tissue of blood in diversepathological states. These novel molecules have great therapeuticpotential in the treatment of gastric ulcers, and inflammatory boweldisease, whereas new agents that inhibit its function could prove usefulin the treatment of cancers of the GI tract.

The stomach is not a congenial location for many bacteria, and thosethat can survive the acidity do not establish themselves there (Rotimiet al., 1990). It is of interest therefore that the antrum region is thefavored site for the attachment, penetration and cytolytic effects ofHelicobaccter pylori, an agent which infects a major proportion of thehuman population (>60% by the seventh decade) and has been associatedwith gastritis, gastric and duodenal ulcers (Goodwin et al., 1986;Blaser, 1987) and gastric adenocarcinomas (Nomura et al., 1991;Parsonnet et al., 1991). Thus as an epithelial cell growth factor,AMP-18 may act to ameliorate the damage caused by bacterial infiltrationand cytolysis. Given the conjunction of the specific antrum expressionof AMP-18 and the preferred site of binding of H. pylori, it is possiblethat the bacteria use AMP-18 as a tropic factor. H. pylori attaches tocells of the antrum having fucose-containing mucin granules (Falk etal., 1993; Baczako et al., 1995). These granules also may containAMP-18. Anti-microbial peptides have been found in the stomach of theamphibian Xenopus laevis (Moore et al., 1991). Some domains of theAMP-18 structure resemble that of the magainins, and possibly AMP-18interacts with enteric bacteria.

6. AMP Peptide 77-97 Inhibits Growth of Human Helicobacter pylori.

To determine if AMP peptide inhibits growth of H. pylori, a lawn ofbacteria was prepared on a culture dish. A small circular filter wasplaced in the center of the dish, a solution of a test agent was placedon the filter so it diffused onto the lawn, and its effect on bacterialgrowth around the filter was measured. AMP peptide 77-97 (SEQ ID NO: 12)(Table 1), a scrambled version of this AMP peptide (negative control)(SEQ ID NO: 19), or the antibiotic clarithromycin (positive control) wasadded to a filter on different cultures. As shown in FIG. 28, thevehicle (control) and the scrambled peptide (negative control) did notinhibit growth of H. pylori, whereas clarithromycin, an agent usedclinically to treat H. pylori infection in humans, and AMP peptide bothinhibited growth of the organism. The growth-inhibitory effect of AMPpeptide appeared to be relatively specific for H. pylori because thepeptide did not alter the growth of the following bacteria and fungi:Staphylococcus aureus, Staphylococcus epidermidis, Enterococcusfaecalis, Streptococcus pneumoniae, Streptococcus agalactiae,Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae,Acinetobacter baumanii, Aspergillus niger, or Candida albicans. Thesefindings suggest that when H. pylori organisms bind to the mucosalepithelial surface of the gastric antrum, its cells could responddefensively by producing and/or secreting full-length AMP-18 form, or apeptide fragment of it that can act as an antibiotic. In addition, AMPpeptide could serve as a therapeutic agent to treat H. pylori infectionsin the stomach, and thereby prevent the capacity of this organism tocause gastritis, gastric ulcers, and gastric adenocarcinomas.

7. Isolation of Pig AMP-18

Antisera against human AMP-18 protein were used to assist in thepurification of the protein from extracts of pig antrum mucosa.Immnoaffinity methods applied to total tissue extracts have not provenvery effective, but by using immunoblots to monitor cell-fractionation,gradient centrifugation and gel electrophoresis, sufficient amounts ofthe pig 18 kDa polypeptide were purified to confirm by sequencing thatthe native N-terminus is the one predicted by cleavage of 20 amino acidsfrom the N-terminus of the ORF precisely at the alanine-aspartate siteanticipated for signal peptide removal. Despite the abundance ofasparagine residues in the mature protein, none fit the consensuscontext characteristic of glycosylation. Fairly extensive regions of theprotein may possess amphipathic helix forming propensity. The latter mayrepresent units within the protein yielding bioactive peptides afterprocessing. Using circular dichroism the synthetic peptide representingamino acids 126-143 in the human preAMP sequence (FIG. 3) is readilyinduced to become helical in moderate concentrations of trifluoroethanolconditions used to assess helix propensity for some bioactive peptides,including anti-microbial peptides of the magainin type (see, forexample, Park et al., 1997).

8. Preparation of Active Recombinant Human AMP-18 in E. coli

A cDNA encoding human AMP-18 was designed in which the 20-amino acidhydrophobic signal peptide sequence was replaced with an N-terminal12-amino acid peptide that included a starch of 6 histidine residues(FIG. 13, left panel). Expression of this modified cDNA sequence waspredicted to yield a 177-amino acid protein product (M_(r) 19, 653) thatcould be readily purified using Ni-NTA resin to bind the His6-tag (SEQID NO: 16). The cDNA sequence lacking the region coding for theN-terminal signal peptide (see FIG. 14) was amplified by PCR usingoligonucleotides that provided suitable linkers for inserting theproduct into the BamH1 site of a QE30 expression vector (QIAGEN); thesequence of the recombinant vector was confirmed. The recombinant human(rh) AMP-18 engineered with the His6-tag (SEQ ID NO: 16) wassubsequently expressed in E. coli cells. To harvest it, the bacteriawere lysed and alquots of the soluble and insoluble fractions weresubjected to SDS-PAGE followed by immunoblotting using the specificrabbit antiserum to the rhAMP-18 precursor. Very little of the expressedprotein was detected in the soluble fraction of the lysate.

Urea (6 M) was employed to release proteins from the insoluble fractionsolubilize rhAMP-18 containing the His6-tag (SEQ ID NO: 16), and make itavailable to bind to the Ni²⁺-charged resin from which it wassubsequently eluted with a gradient of imidazole (0 to 200 mM). Theamount of eluted rhAMP-18 was measured using the BCA assay, and theappearance of a single band at the predicted size of 19-20 kD wasconfirmed by SDS-PAGE followed by immunoblotting. To determine if elutedrhAMP-18 renatured to assume a structure that was mitogenic, aliquots ofthe eluate (following removal of urea and imidazole by dialysis) wereadded to cultures of IEC-18 cells and the number of cells was counted 4days later. FIG. 13 (right panel) indicates that the recombinant proteinstimulates cell proliferation to the same maximal extent as doesmitogenic AMP peptide 77-97 (or soluble antrum tissue extracts from pigshown in FIG. 11), but that it does so at a half-maximal concentrationan order of magnitude lower than for peptide 77-97. AMP peptide 77-97refers to the mature protein; same as peptide 97-117 of human precursorprotein: Table 1. These observations indicate that biologically activerecombinant human AMP-18 that can be utilized in diverse clinicalsituations is available. The mitogenic potency of rhAMP-18 is in thenanomolar range which would be expected for a native gastric cell growthfactor that participates in the maintenance and repair of the stomach invivo.

The demonstration that amino acids 77-97 represent a functional domainof AMP-18 suggest that the full-length protein could easily be modifiedat its N- and/or C-terminus. Targeted modifications could prolong thehalf-life of AMP-18 in the circulation and tissues in vivo, therebyenhancing its pharmacokinetic profile without adversely affecting itsdiverse biological functions.

9. Stimulation of Growth and Restitution of Stomach and IntestinalEpithelial Cells by AMP-18 and Derived Peptides

To characterize the capacity of gastric and intestinal cells to respondto AMP-18 , AGS gastric adenocarcinoma cells, HAE human gastric antrummucosa primary cultures transformed with SV40 large T antigen, ratdiploid small intestinal epithelial cells of the IEC-6 (FIG. 15) andIEC-18 lines, NCI N-87 gastric carcinoma cells, and SK-GT5gastroesophageal adenocarcinoma cells were studied; human WI-38fibroblasts and HeLa cells served as non-GI control cell lines.Mitogenesis was assayed by performing cell counts 3 to 4 days afterexposing cells to the agent of interest, trypsinizing the culture toprepare single cells, and confirming this while counting them in ahemocytometer.

Antrum extracts containing AMP-18 , peptide 77-97, or EGF eachstimulated growth of AGS cells, and as expected, the rabbit antiserum torecombinant human AMP-18 precursor protein inhibited the activity of theantrum extract but not of peptide 77-97 which lacks the epitope (FIG.11). Growth stimulation by peptide 77-97 was additive with that of EGF.Growth of AGS cells is not stimulated by scrambled peptide 77-97 or bypeptide 67-85, and peptide 67-85 completely inhibits growth stimulationby peptide 58-99. HAE cells were used to test whether AMP-18 can exertan effect on epithelial cells that exist in he local environment of itssynthesis. These cells, provided by Dr. Duane Smoot, Howard UniversityCollege of Medicine, are not completely immortalized and therefore havelimited passage number. Growth stimulation of HAE cells by peptide 77-97was apparently additive with that of EGF (FIG. 16, left panel). Not onlydoes the AMP peptide stimulate growth but it also acted as a motogen,resulting in more rapid migration (restitution) of cells into scrapewounds made in confluent cultures. This enhancement of wound restitutionalso showed high additivity with EGF (FIG. 16, right panel). Whetherthere is a synergism or not, the observed additivity supports thatAMP-18 may play an important role in maintaining an intact stomachmucosal epithelium, and in facilitating its repair after injury. Thegrowth of rat diploid IEC-6 cells was also stimulated by the antrumextract, peptide 77-97, and EGF, although the peptide appeared a morepotent mitogen than EGF (FIG. 15). Near-maximal growth stimulation wasdetected at an AMP peptide concentration of 0.5 μg/ml (0.23 μM) (FIG.15, center panel), a much lower value than the concentration needed fortrefoil peptides (1 μg/μl) (˜150 μM) or the α-defensin, cryptdin 3 (660μm/ml) (˜140 μM) to exert their effects in culture. The maximalmitogenic effect of rhAMP-18 on IEC-18 cells has been observed at 5nanomolar (FIG. 18). The mitogenic effect of peptide 77-97 wascorroborated by measuring [³H]thymidine incorporation into DNA in IEC-6cells which was stimulated by 68% (P<0.001) from 16,668±616 to28,036±882 by the peptide. Stimulation of wound restitution wascomparable to EGF, and apparently additive with it. Scrambled peptide77-97 did NOT stimulate growth of IEC-18 cells or BSC-1 cells atconcentrations up to 8 82 g/ml. Growth of gastric NCI N-87 cells andgastric SK-GT5 cells was also stimulated by peptide 77-97, antrumextract, of EGF in a concentration-dependent manner. AMP-18 antiserumblocked the mitogenic effect of antrum extract, or EGF in aconcentration-dependent manner. AMP-18 antiserum blocked the mitogeniceffect of antrum extract on these two gastric epithelial cell lines, butnot the proliferative effects of peptide 77-97 or EGF. Preimmune serumhad no effect on growth. These results show that AMP-18 and its peptidederivatives could function in vivo to stimulate growth and restitutionduring repair after injury.

The failure of AMP peptide to stimulate growth of human of fibroblastic(WI-38) or epidermoid (HeLa) cells at concentrations up to 8 μg/mlsuggests that the mitogenic effect of the peptide is epithelial-cellspecific.

10. Competitive Inhibition of IEC-18 Cell Growth by AMP-derived Peptides

To gain additional information about the interaction between AMPpeptides and their binding site(s) on the cell surface, non-transformedrat IEC-18 cells were studied. Progressively increasing theconcentration of non-mitogenic peptide 67-85 blocks growth-stimulationby peptide 58-99 if this mitogenic 42-mer exerts its effect by areceptor-mediated mechanism. Peptide 58-99 stimulated an increase incell number of 407% compared to 290% by the vehicle in a 3-day assay. Asthe concentration of peptide 67-85 was raised progressively to ˜0.1μg/ml, the growth-stimulatory effect of peptide 58-99 was nearlyabolished (FIG. 17). This result shows that the two peptides compete forthe same surface “receptor” site.

11. Antiserum to AMP-18 Neutralizes the Mitogenic Effect of rhAMP-18

Rabbit antiserum to AMP-18 precursor recognizes rhAMP-18 on immunoblots.The antiserum also blocks the mitogenic effect of porcine antral tissueextracts (FIG. 11) and AMP peptide 58-99, and immunolocalizes AMP-18 incells of human and murine gastric antral tissue. FIG. 18 shows that theantiserum neutralizes the mitogenic effect of rhAMP-18 in confluentcultures of IEC-18 cells, thereby extending its utility to study therecombinant as well as native protein. Although AMP peptide 77-97requires a relatively higher molar concentration to exert its mitogeniceffect than does rhAMP-18, (FIG. 13), this result also indicates thatthat AMP peptide is an appropriate surrogate for rhAMP-18.

To improve the yield of rhAMP-18, an EDTA-free protease-inhibitorcocktail is used, lysozyme is added to digest E. coli cell debris, andrecombinant protein is eluted from Ni²⁺beads with 1M imidazole.

12. AMP Peptide Stimulates Restitution of Gastric and IntestinalEpithelial Cells after Scrape-wounding

Data presented in FIG. 19 were obtained after 24 to 48 hr exposure toAMP peptide, times before a mitogenic effect can be detected by anincrease in cell number. The results indicate that AMP peptidestimulates restitution in scrape-wounded human gastricadenocarcinoma-derived cells of the HAE line, and in nontransformed ratintestinal cells of the IEC-18 line. Thus AMP peptide rapidly stimulatesrestitution of gastric and intestinal epithelial cells in culture, andis expected to speed resurfacing of the injured gastric mucosa in vivo.

13. Mitogenic and Motogenic Effects of AMP Peptide in Cell CultureSupport a Therapeutic Role in Gastric Mucosal Injury

The synthesis of AMP-18 is confined to lumenal mucosal lining epithelialcells of the gastric antrum of humans and other mammals. Inside cellsthe protein is co-localized with mucins in secretion granules, andappears to be secreted into the mucus overlying the apical plasmamembrane. Recombinant human AMP-18 prepared in E. coli exerts itsmitogenic effect at a concentration an order of magnitude lower thangrowth-promoting peptides derived from the center of the mature protein.Peptide 77-97, the most potent mitogenic peptide, is amino acidsequence-specific, and appears to be cell-type specific as it does notstimulate growth of fibroblasts or HeLa cells. Mitogenesis by specificAMP peptides appears to be mediated by a cell surface receptor becausecertain peptides that are not active mitogens can competitively inhibit,in a concentration-dependent manner, the growth-stimulating effects ofpeptide 58-99 and antrum cell extracts. AMP-18 and its derived peptidesexhibit diverse effects on stomach and intestinal epithelial cellsmaking it likely they play a critical role in repair after gastricmucosal injury. These include mitogenesis, restitution, cytoprotection,and maturation of barrier function after indomethacin-mediated injury.Certain of these physiological effects can occur at concentrations thatare relatively low for rhAMP-18 (<50 nM) compared to the concentrationsof other gastric peptide mediators such as trefoil peptides or theα-defensin, cryptdin 3 (>100 μM). Immunoreactive AMP-18 is apparentlyreleased by cells of the mouse gastric antrum after indomethacin gavage,and by canine antrum cells in primary culture exposed to forskolin,suggesting that the protein is subject to regulation. AMP-18 likelyplays a role in physiological and pathological processes such as woundhealing in the gastric mucosal epithelium in vivo as may occur ingastritis secondary to non-steroidal anti-inflammatory drugs, otherpharmaceutical agents and alcohol, ulcer disease, and the consequencesof H. pylori infection and inflammation.

14. AMP Peptide 77-97 Enhances Development of Barrier Function ofEpithelial Cells and is Cytoprotective

Maintenance of barrier function is essential for preventing entry offoreign antigens and bacteria from the gastric lumen, and for otherfunctions such as vectorial transport of electrolytes, water andnutrients. Acting alone or in concert with other agents, AMP-18 mediatesthe rapid return of barrier function following mucosal injury. Todetermine whether AMP peptide 77-97 could facilitate development ofbarrier function, and could also serve as a cytoprotective agent toprevent loss of function when reactive oxygen metabolites, indomethacin,or dextran sulfate sodium (DSS), increases mucosal permeability andcompromises cell integrity needed to maintain epithelial tightjunctions. Cell lines known to develop relatively high values for TER asa marker of epithelial tight junctions were used. Initially, peptide77-97 modulates maturation of TER in monolayer cultures ofwell-characterized, nontransformed MDCK cells. FIG. 20 shows exposure tothe peptide increases TER in the monolayer by 24 hr, and to a greaterextent thereafter. This observation suggests that AMP-18 or AMP peptidespeeds recovery of the GI epithelium after injury, and enhancesdevelopment of barrier function.

To determine whether AMP peptide protects barrier function in a tissueculture model of mucosal oxidant injury, cell monolayers were subjectedto reactive oxygen metabolite injury using monochloramine. The resultsin FIG. 21 (panel A) indicate that after 60 min of exposure tomonochloramine, MDCK cells treated with vehicle or EGF show asubstantial loss of TER, whereas the TER of cultures treated withpeptide 77-97 is similar to non-injured monolayers. These results are ofconsiderable interest because they indicate that AMP peptide but not EGFis cytoprotective under this set of conditions, whereas these twomolecules were previously found to be equivalent and additive mitogensand motogens for gastric and intestinal epithelial cells. Thecytoprotective effect of peptide 77-97 was also apparent in Caco2/bbe(C2) cells derived from a human colonic adenocarcinoma line in thesetting of oxidant (FIG. 22, panel B) or indomethacin-mediated (panel C)injury.

15. AMP Peptide Protects Against DSS-mediated Injury of Cells inCulture, and also Speeds Recovery of TER and Restitution After Injuryhas Occurred

To evaluate the potential capacity of AMP peptide to exert acytoprotective effect in colitis in vivo, a solution of dextran sulfatesodium (DSS) was added to the culture medium of C2 cell monolayers usedto model the colonic epithelium. DSS-mediated injury of barrier functionwas quantified be measuring TER in these monolayer cultures. FIG. 22indicates that DSS (4%) reduced the TER to ˜30% of the control valueafter 45 min, and that AMP peptide was cytoprotective. This observationprovides a strong physiological rationale for evaluating AMP peptide asa therapeutic agent in the murine model of DSS-mediated colitis.

To determine whether AMP peptide could speed recovery of TER afterDSS-induced colonic cell injury, a highly sought-after functionalcharacteristic of an agent designed to treat IBD, C2 cell monolayerswere exposed to DSS (5%) for 10 min which reduced TER to 33±6% of thecontrol value (FIG. 29, left panel). DSS was removed by aspirating themedium and replacing it with fresh medium. AMP peptide 77-97 (8 μg/ml)or vehicle was added to the culture medium, and TER was measured 18 hrlater. In the presence of the vehicle, TER increased from 33% to 66±7%of the control value, whereas cells exposed to AMP peptide reached avalue 112±4% of control. The salutary results in a tissue culture modelof DSS-mediated colitis suggest that AMP peptide can speed recovery ofbarrier function in the injured colonic epithelium in vivo.

In a separate set of experiments DSS reduced restitution ofscrape-wounded cultures of non-transformed diploid rat intestinalepithelial IEC-18 cells (P<0.001) (FIG. 29, right panel). When DSS wasremoved by aspirating the medium and the cultures allowed to recover for24 hr after scrape wounding, cell migration during the next 24 hr was40% greater in the presence of AMP peptide compared to vehicle(P<0.001).

16. The Cyprotective Effect of AMP Peptide in Colonic Epithelial Cellsmay be Mediated by Increased Accumulation of Tight Junction Proteins

FIG. 21B shows that AMP peptide 77-97 blunts the fall in transepithelialelectrical resistance (TER) in Caco2/bbe (C2) cells after oxidantinjury. To find out how the peptide exerts its cytoprotective effect, C2cell monolayers were treated with AMP peptide, and oxidant injury wasinduced with monochloramine 18 hours later. Changes in the levels ofspecific tight junction (TJ) proteins were checked. Cells were lysed,and proteins of the insoluble/particulate fraction were studied byimmunoblotting. FIG. 23 shows that there is more immunoreactive ZO-1 andoccludin in AMP peptide-treated than in vehicle-treated cells at time 0,and for 60 minutes following oxidant-induced injury, showing that thegreater abundance of these TJ proteins thereby blunts loss of TER in themonolayer, preserves barrier function, and could thereby mediate AMPpeptide's cytoprotective effect. This observation represents the firstevidence that AMP peptide enhances accumulation of specific TJ proteinsin colonic epithelial cells. This result implies that AMP peptideenhanced TJ protein accumulation during the 18 hours before cells weresubjected to oxidant injury. Non-injured cells were studied and showedthat AMP peptide (or rhAMP-18) rapidly increased the amount ofimmunoreactive occludin and ZO-1 compared to untreated cells (FIG. 24).To carry out this experiment, C2 cells were exposed to AMP peptide fordifferent periods of time, the NP-40-insoluble fraction was obtained,Western blots were prepared, and the amount of each immunoreactiveprotein was quantified. A 65% increase in immunoreactivity of eachprotein compared to control was detected for occludin after 2 hr, andfor ZO-1 at 8 hr. Hsc73, which is constitutively expressed in thesecells, was not altered by exposure to AMP peptide for upto 18 hr.

To determine if the apparent increase in occludin immunoreactivityinduced by AMP peptide in control cells and those subjected to oxidantinjury was localized to the TJ, confocal microscopy was performed on C2cell monolayers. FIG. 30 shows that the increased occludinimmunoreactivity in control and oxidant-injured cells observed byimmunoblotting (FIG. 23), is largely localized to cell junctions,presumably to the TJ. Similar results using confocal microscopy wereobtained for ZO-1. Taken together, these results show that AMP peptideincreases accumulation and recruitment of occludin in colonic epithelialmonolayers both under control conditions and following oxidant injury,suggesting a mechanism whereby the peptide could exert itscytoprotective effect on barrier function (FIG. 21). Next it wasdetermined if the capacity of AMP peptide to increase accumulation of TJproteins was peptide-specific. FIG. 31 shows that AMP peptide-mediatedaccumulation of occludin and ZO-1 was not replicated when C2 cellmonolayers were exposed to vehicle, EGF, or the scrambled AMP peptideGKPLGQPGKVPKLDGKEPLAK).

Because these studies showed that AMP peptide augmented the accumulationof at least two TJ proteins, this effect was characterized in greaterdetail. Confluent monolayers of C2 cells were treated with AMP peptideor vehicle for 8 hr. Cells were then collected, proteins in theNP-40-insoluble fraction were separated by SDS-PAGE, immunoblots wereprepared, and the relative amounts of immunoreactive proteins werecompared by densitometric analysis. The results in FIG. 32 showincreased accumulation (3- to 6-fold) of the TJ-associated proteinsoccludin, ZO-1, ZO-2, and claudin-5, and the adherens junction proteinE-cadherin (2-fold) in cells treated with AMP peptide. In addition, a˜50% increase in accumulation of junctional adhesion molecule (JAM),another TJ protein, was observed at 48 and 72 hr after exposure to AMPpeptide compared to control (0 hr) (FIG. 33). These changes appearrelatively specific for occludin, ZO-1, ZO-2, claudin-5, E-cadherin, andJAM because they were not observed for several other proteins localizedin the TJ (claudin-1, claudin-2), adherens junction (β-catenin), plasmamembrane (Na-K-2Cl cotransporter, α or β subunit of Na-K-ATPase), orcytosol (Rho A, hsc73).

Taken together, these results suggest that AMP peptide could exert itscytoprotective effect in colonic epithelial cell monolayers both byincreasing accumulation of specific tight and adherens junctionproteins, and protecting against their loss at the time of injury.

17. Cell Surface Specificity of AMP Peptide-stimulated Accumulation ofTJ Proteins

To better understand the mechanism(s) by which AMP peptide enhancesaccumulation of TJ-associated proteins, experiments were performed todetermine whether the peptide exerted its effect at the apical orbasolateral surface of C2 cells. Monolayers of C2 cells were grown onTranswell filters, AMP peptide (8:g/ml) was added to the apical orbasolateral compartment of the Transwell, and cells were harvested 8 hrlater. The NP-40-insoluble fraction was obtained, its proteinsseparated, and immunoblots were prepared. FIG. 34 shows that exposure ofthe basolateral but not the apical plasma membrane to AMP peptide wasassociated with increased accumulation of occludin and claudin-5, butnot hsc73. These results suggest that receptors for AMP peptide resideprimarily on the basolateral rather than the apical surface of colonicepithelial cells.

18. AMP Peptide Prevents Loss of Occludin in C2 Cells

The report that occludin protein and mRNA are reduced in colonic tissueof patients with IBD (Kucharzik et al., 2001) focused our attention morerigorously on this TJ protein in C2 cell monolayers used to model thecolonic mucosal surface. To identify the high molecular weightphosphorylated form of occludin that is found within TJs at the apicalplasma membrane, 12.5% polyacrylamide gels were used (Wong, 1997). Twomajor forms of occludin in the NP-40-insoluble fraction of C2 cellsgrown on plastic dishes were correlated with the TER of monolayercultures grown on Transwells. Between 1 and 15 days after plating, TERincreased from 132 to 278 Σ≅cm ², and total occludin appeared toincrease nearly 5-fold (as assessed by densitometric analysis) (FIG.35). When C2 cells were exposed to DSS (4%) which markedly reduces TER(FIG. 22), total occludin immunoreactivity declined by about 50% at 1 hr(FIG. 36, top, right panel) compared to vehicle-treated control cultures(top, left). When control cells were treated with AMP peptide (8:g/ml)for 18 hr, occludin immunoreactivity doubled (bottom panel, left)compared to vehicle-treated control cells (top, left), and when AMPpeptide-treated cells were subsequently exposed to DSS, occludinimmunoreactivity did not decline (bottom, right).

Purified rhAMP-18 was also tested to determine if it blunted the fall inTER in C2 cells following oxidant injury. FIG. 25 shows that exposure tomonochloramine reduces TER by ˜35% at 45 min, whereas cells pre-treatedwith either rhAMP-18 or on an AMP peptide exhibited only a ˜10% declinein TER. FIG. 26 indicates that treatment of C2 cells with rhAMP-18 for 8hr increases the amount of immunoreactive occludin and ZO-1 compared tovehicle-treated cells.

These experiments show that injuries of C2 cell monolayers inflicted byDSS or monochloramine reduce both TER and immunoreactive occludin, andthat pretreatment of the cells with AMP peptide as well as rhAMP-18exerts a cytoprotective effect that prevents both the loss of occludinand decline in TER. AMP-18 likely mediates its cytoprotective effect byenhancing accumulation of specific TJ proteins and thereby preservingbarrier function along the GI tract in the setting of mucosal injury.

19. AMP Peptide Protects Barrier Function Following Disruption of theAct in Filament Network in C2 Cells

To determine if the cytoprotective effect of AMP peptide is mediated atthe level of the actin cytoskeleton, C2 cell monolayers were exposed tocytochalasin D, an agent known to disrupt actin filaments and compromisebarrier function (Madara et al., 1986). Exposure of the mature cellmonolayer to a low concentration of cytochalasin D (0.01 μg/ml)progressively reduced the TER by 34% (P<0.001) (n=9 cultures) comparedto vehicle after 2 hr (FIG. 37). The TER of cells pretreated with AMPpeptide (8 μg/ml) for 18 hr prior to exposure to cytochalasin D did notdecline significantly after 2 hr (n=11), and was higher thanvehicle-treated cells (P<0.001). The capacity of AMP peptide to preventcytochalasin D-induced barrier dysfunction suggests that it may exertits cytoprotective effect at the level of the actin cytoskeleton.Confocal microscopy revealed that treatment of the cells with AMPpeptide protected perinjunctional actin in the setting of oxidant (0.3mM monochloramine) injury.

20. Administration of AMP Peptide Delays appearance of Blood in theStool and Reduces Weight Loss in Mice with DSS Induced Colitis

To evaluate the therapeutic efficacy of AMP peptide in vivo, colitis ofmild to moderate severity was induced in C57BL/6 male mice by giving theanimals DSS (3%) dissolved in tap water to drink ad libitum. Evidence ofcolitis (blood in the stool assayed by hemoccult strips) was found asearly as day 1 (FIG. 27, left panel), and in all animals giveninjections of the vehicle subcutaneously (s.c.) by day 4. AMP peptidewas administered s.c. one day before animals were given DSS, and onceper day thereafter. Bloody stool was detected in fewer animals given AMPpeptide than the vehicle (P<0.01; n=50).

To look for a systemic effect of treatment with AMP peptide during thedevelopment of DSS colitis, body weight was measured. During the first 4days of DSS administration, body weight changed little in peptide- orvehicle-treated mice. Then DSS was discontinued, the animals were givenwater to drink (day 0; FIG. 27, right panel), and measurements of weightwere continued. During the next 3 days, mice given a daily injection AMPpeptide lost less weight (P<0.01) than animals given the vehicle. By day7 after stopping DSS, the weight gain of mice given AMP peptide resultedin complete recovery of weight that was lost, whereas animals given thevehicle continued to lag behind (P<0.01). To better understand how AMPpeptide could exert these salutary effects on hematochezia and weightloss in DSS colitis, studies were performed to determine if observationsin cultures of C2 cells were also relevant in vivo. Because DSStreatment appeared to injure barrier function in monolayer cultures ofC2 cells, assessed as a decrease in TER (FIG. 22) and reduced occludinin the detergent-insoluble fraction (FIG. 36), a question was whetherDSS might adversely affect the content of this TJ protein in vivo. Mice(n=10) were given DSS (3%) or water to drink. Four days later animalswere killed, the contents of the colon lumen including blood were washedout, and the mucosa was inspected. The colonic epithelial surface ofmice given DSS appeared largely intact with no obvious ulcerations ordenuded areas. Surface cells of visibly intact mucosa were thencollected by scraping with a glass slide. Occludin levels in theNP-40-insoluble fraction were assessed by immunoblotting. FIG. 38 showsthat DSS administration resulted in a 50% decline (P<0.01) in occludinimmunoreactivity in mucosal cells compared to mice given water to drink.In contrast, when probed with antisera to β-catenin and hsc73, blotsfrom mice given DSS or water to drink displayed equivalent levels ofimmunoreactivity, suggesting that the difference in FIG. 38 was not theresult of a smaller amount of epithelial cells in scrapings obtainedfrom DSS-treated mice than those drinking water. These results presentone way in which DSS colitis in mice appears similar to IBD in humans:in both mice (FIG. 38) and humans (Kucharzik et al., 2001), colitis isassociated with a decline in colonic mucosal occludin content, althoughthe mechanisms that mediate the disease in each syndrome are known to bedifferent. These findings appear to validate the DSS mouse as a model ofcolitis.

Next, experiments were performed to determine if AMP peptide couldstimulate accumulation of occludin in the mouse colonic mucosa in vivo,as was observed in human colonic epithelial cells in culture (FIGS. 23,24). Control mice were injected (s.c.) daily with 10, 20, or 30 mg/kg ofAMP peptide for 2 to 6 days. Occludin immunoreactivity in colonicsurface cells was studied with a protocol like that used to obtain theresults presented in FIG. 38. Some but not all mice showed an increasein occludin content by day 2, and nearly all on day 4. FIG. 39 showsthat treatment for 5 days with AMP peptide (n=3 mice) increased theamount of immunoreactive occludin in the NP-40-insoluble fraction by˜2-fold compared to vehicle-treated mice (n=3) (P<0.4), and that bothhigh molecular weight (presumably hyperphosphorylated) occludin, as wellas the lesser or nonphosphorylated forms were more abundant (P<0.004).Treatment with AMP peptide appeared to be relatively specific, as it didnot alter the amount of the adherens junction protein β-catenin, as wasalso the case when C2 cells were treated with the peptide.

21. AMP Peptide is Cytoprotective in C2 Cell Monolavers Exposed toPseudomonas aeruginosa-I Lectin, and Prevents Death in Pseudomonasaeruginosa-induced Gut-derived Sepsis in Mice

Observations in cell culture and in mice with DSS-induced colitisdisclosed herein show that treatment with AMP peptide likely stabilizestight junctions in the gut and thereby prevents the lethal effect ofgut-derived sepsis in mice.

The presence of Pseudomonas aeruginosa (PA) in the intestinal tract ofcritically-ill patients is associated with a 70% death rate which is3-fold higher than in age-matched critically-ill subjects who havenegative cultures for this organism. In both monolayer cultures of humancolonic (C2) cells and in living mice, the bacterial surface lectin PA-Ibehaves as an adhesin that binds to the apical cell surface (Laughlin etal., 2000). When PA-I glycoprotein, or living bacteria were added to C2cell monolayer cultures, the TER declined and the amount of the TJproteins occludin and ZO-1 fell. Injection of living P. aeruginosa intothe cecum of mice subjected to stress (30% hepatectomy and fooddeprivation postoperatively) was consistently lethal for the animals(Laughlin et al., 2000). These cell culture and mouse models ofgut-derived sepsis (GDS) suggest that P. aeruginosa, through its adhesinPA-I, binds to the surface of colonic epithelial cells therebydisrupting TJ proteins (occludin and ZO-1) and induces a permeabilitydefect that permits entry of the P. aeruginosa exotoxin A to breech themucosal barrier. The translocation of exotoxin from gut lumen into thesubmucosal compartment and then into the circulation would subsequentlykill the mouse.

To determine if AMP peptide could play a cytoprotective role in GDS, astudy was carried out in C2 cell monolayers that were exposed to thePA-I lectin. The TER of cells exposed to PA-I lectin declined by ˜22% at4 hr when treated with the vehicle, whereas monolayers treated with AMPpeptide decline 0-5% at that time, suggesting a cytoprotective effect(FIG. 40). Next the effect of AMP peptide was studied in the mouse modelof P. aeruginosa-induced-GDS (Laughlin et al., 2000) using female Balb/cmice subjected to 30% hepatectomy, injection of a solution of living P.aeruginosa into the cecum, and food deprivation postoperatively. All 8of the mice survived surgery. One day later, all 4 animals pretreatedwith the vehicle (s.c.) were dead, whereas each of 4 mice treated withAMP peptide (15 mg/kg i.p for 4 days, s.c. for 1 day) were alive andwell (FIG. 41). These observations in cell culture and in vivo suggest atherapeutic rationale for using AMP peptide to treat GDS in humans.

22. AMP Peptide Increases Accumulation of Heat Shock Proteins

In an effort to identify molecules that could mediate the cytoprotectiveeffect of AMP peptide, it was asked if exposing intestinal epithelialcells to the peptide could modify induction of heat shock proteins(hsps) (Musch et al., 1996). Diploid non-transformed rat intestinalepithelial IEC-18 cells were used to study induction of hsps in responseto thermal injury rather than C2 cells—which constitutively expresshsp27 and hsp72 at high levels, possibly because they are transformed.(Hsp27 is found in human C2 cells, and hsp25 in rat IEC-18 cells. Hsp72is often referred to as hsp70). The results depicted in FIG. 42 indicatethat, as expected, hsc73 and hsp25 are constitutively expressed (0 hr),but hsp72 is not. Monolayer cell cultures were exposed to AMP peptideimmediately after heat shock injury. Twelve hours later, expression ofboth hsp25 and hsp72 was greater in the peptide-treated thanvehicle-treated cultures. An increased amount of both hsps persisted forat least 24 hr in peptide-treated cells, but declined to basal values by48 hr. These results suggest that the cytoprotective effect of AMPpeptide could be mediated, at least in part, by increased accumulationof stress proteins that protect the integrity of the actin cytoskeletonand help maintain TJs, thereby defending mucosal barrier function asdescribed in other types of cells (Lavoie et al., 1993).

23. AMP Peptide Induces Tyrosine Phosphorylation

To find signaling molecules by which AMP peptide mediates its biologicaleffects, it was asked if the peptide stimulates tyrosine phosphorylationin GI epithelial cells. IEC-18 cells were treated with AMP peptide fordifferent periods of time, the cells were then lysed, the proteinsextracted and separated on SDS-polyacrylamide gels, blotted, and theblot was probed with 4G10 anti-phosphotyrosine monoclonal antibody.Exposure of cells to AMP peptide resulted in tyrosine phosphorylation ofseveral proteins after two min, suggesting that the peptide stimulatestyrosine kinase activity upon its interaction with the cell surface(FIG. 43). There was a decline in the extent of tyrosine phosphorylationof several proteins after 5 min, and persistence of others for up to 60min. These observations suggest that protein tyrosine phosphorylationplays a role in the mechanism(s) by which AMP peptide mediates itscytoprotective, mitogenic, and motogenic effects.

24. AMP Peptide Activates p38 MAP Kinase and Induces Phosphorylation ofhsp25

AMP peptide increases accumulation of hsp25 in cells stressed by heat(FIG. 42), and may stabilize the actin cytoskeleton following exposureto the actin-disrupting agent, cytochalasin D (FIG. 37). Because AMPpeptide appears to exert its cytoprotective effect on both hsp25 andactin, additional evidence was sought to determine if the peptide couldactivate the p38 MAPK/hsp27/actin filament pathway (Lavoie et al.,1995).

First the effect of AMP peptide on phosphorylation of p38 MAPK in IEC-18cells was examined. After exposure to the peptide (8 μg/ml) for up to 2hr, the cells were lysed, the proteins were extracted, and Western blotanalysis was performed using anti-phospho-specific p38 antibody. Thenthe membrane was stripped and reprobed with anti-p38 antibody. FIG. 44(left panel) reveals that AMP peptide resulted in a striking increase inp38 phosphorylation at 30 min, with no change in total p38 MAPK. Next,to see if AMP peptide stimulated phosphorylation of hsp25, a specificanti-phospho-hsp25 antibody (Affinity BioReagents) was used to probe ablot prepared using AMP peptide-treated IEC-18 cells as above. Treatmentwith AMP peptide stimulated the appearance of phospho (P)-hsp25 at 30min, and was also associated with an increase in total hsp25 after 1 hr(FIG. 44, right panel).

Taken together with observations disclosed herein, these results suggestthat AMP peptide activates the p38 MAPK/hsp27/actin pathway. Thesefindings are also consistent with the hypothesis that an early effect ofAMP peptide-induced stimulation of hsp25/27 phosphorylation mediatesentry of P-hsp25 into the nucleus (Geum et al., 2002) wherein itfunctions as a transcriptional activator, whereas at later times,P-hsp25 may act to stabilize the actin cytoskeleton by binding to themicrofilaments (Huot et al., 1996). Because AMP peptide also stimulatesmitogenesis and motogenesis, a question was whether if the JNK and ERKpathways were also activated. FIG. 45 shows that both JNK1/2 and ERK1/2appear to be activated by AMP peptide, as indicated by the rapidappearance of phosphorylated JNK and ERK.

25. AMP Peptide Activates Protein Kinase C Zeta

To learn more about how AMP peptide could participate in theestablishment of TJ structures and epithelial cell polarity duringepithelial wound healing, attention was focused on protein kinase (PK) Czeta (ζ), an atypical PKC that appears to be required for assembly ofTJs (Suzuki et al., 2001). PKCζ, presumably in its active formphospho-PKCζ, regulates TJs, apparently by interacting with severalTJ-associated proteins including occludin, PAR-6, and PAR-3. In C2cells, AMP peptide stimulated an increase in phosphorylation of PKCζwithin 20 min when studied by immunoblotting. This was confirmed andextended with confocal microscopy which showed accumulation ofphospho-PKCζ in the cell cytosol 20 min after exposure to AMP peptideand its subsequent translocation to the TJ at 60 min. These findingsshow that AMP peptide acts to establish new TJ structures duringepithelial wound healing after cell injury by activating(phosphorylating) PKCζ.

Materials and Methods

1. Isolation of Antrum-specific cDNA Clones

cDNA clones for the gastrointestinal peptide gastrin, which regulatesgastric acid secretion as well as mucosal and pancreatic cell growth(Yoo et al., 1982) were isolated. From these screens several other mRNAsexpressed relatively specifically in the antrum of the stomach werefound. The open reading frame (ORF) in one of these RNAs was highlyconserved between pig and man, and predicted a novel conserved proteinof no immediately apparent function. Using specific antibodies, it wasshown that similar protein species are present in the stomach antrummucosa of all mammals tested. There is tissue specificity of expressionof these sequences and they are apparently ubiquitously present in theantrum mucosa of mammalian species.

2. RNA Expression

The isolation of the cDNA clones was predicted on a preferentialexpression in the mucosa of the stomach antrum and this has beenconfirmed initially by Northern blot hybridization of RNAs from varioustissues probed with the cDNA sequences and subsequently by proteinanalysis. The Northern blots showed the specificity of mRNA expressionwithin the gastrointestinal tract of the pig. Highest mRNA expressionwas in the antrum mucosa, variable amounts in the adjacent corpus mucosaand undetectable levels in fundus, esophagus and duodenum. Thenon-mucosal tissue of the antrum and corpus contained little RNAreacting with the cDNA probe.

3. Antibodies to Expressed Protein

The open reading frames (ORFs) of the human and pig cDNA clones predictvery similar relatively low molecular weight (MW) proteins, which haveno close homologs to known proteins in the computer databases andtherefore give little indication of possible function. As an approach tostudy the biological role of the presumptive proteins, the full cDNAsequences were expressed in E. coli, using a vector that also encoded anN-terminal His6-tag (SEQ ID NO: 16). Unfortunately, as expressed inbacteria the polypeptide products are insoluble and not readily amenableto biochemical studies. However, the bacterial product of the human cDNAwas separated on sodium dodecyl sulfate (SDS) gels used as an immunogenin rabbits to elicit antisera. The sera were screened against proteinextracts of antral tissue from a number of mammalian species. Thisprocedure has successfully produced several high-titer, low backgroundantisera capable of recognizing both the immunogen and proteins of about18 kDa expressed in the antrum of the mammals tested. Thebacterially-expressed protein migrates more slowly because it containsthe signal peptide sequence was well as a His6-tag (SEQ ID NO: 16). Thepreimmune sera showed no significant 18 kDa reactivity. Thecross-reactivity of the antisera raised against the protein expressedfrom the human cDNA clone with proteins of very similar MW in antrumextracts from a variety of mammals (pig, goat, sheep, rat and mouse; thelast consistently migrates slightly more rapidly in SDS gels) supportsthe level of conservation of amino acid sequence predicted by comparisonof the ORFs of the human and pig cDNAs (see FIG. 10). In subsequentexperiments, human AMP-18 with a signal peptide was produced inbacteria.

The preimmune sera give insignificant reactions on Western blots of alltissue extracts, while the two immune sera (at up to 1:50000 dilution)both give major bands of 18-20 kDa only, and those only in stomachantrum extracts, and to a lesser degree in the adjacent corpus extracts.The sera were raised against bacterially-expressed protein so there isno possibility of other exogenous immunogens of animal origin.

As determined by immunoblots, the specificity of expression to theantrum is even greater than the Northern blots would suggest, and thestrength of the signal from antrum extracts implies a relatively highabundance of the protein, although quantitative estimates were not made.Significant antigen was not detected in non-stomach tissues tested.

The immunohistochemistry showed insignificant staining of antral tissueby both preimmune sera, while both immune sera stained the surfacemucosal cells very strongly at considerable dilutions. The preimmunesera did not lead to immunogold staining in the immunoelectronmicroscope study. The growth factor activity of antrum extracts isinhibited by both immune, but not preimmune sera. Finally, the resultswith a synthetic peptide, which has growth factor activity, is inhibitedby the immune but not the preimmune sera, and carries epitopesrecognized by the immune but not the preimmune sera, further validatethe specificity of these reagents.

4. Northern Blot Hybridization of RNAs from Pig Gut Mucosal Tissues

Total RNA was electrophoresed, transferred to a membrane and hybridizedwith a labeled pig AMP-18 cDNA probe. The source of the RNA sample foreach lane was: distal duodenum, proximal duodenum, antrum, adjacentcorpus, fundus, and esophagus. Equal amounts of RNA were loaded. Thesignal from RNA of the antrum adjacent corpus was variable. Size markers(nucleotides) were run on the same gel for comparison.

5. Immunoblots Using a Rabbit Antiserum Raised Against theBacterial-expressed Protein Directed by the Human Antrum-specific cDNAClone

Whole tissue proteins were dissolved in SDS buffer, electrophoresed, andtransferred to membranes that were reacted with immune serum (1:50000).Bound antibody molecules were detected using peroxidase-labeledanti-rabbit antibody. Preimmune serum gave no specific staining ofparallel blots at 1:200 dilution.

Immunoblots of high percentage acrylamide gels showed that the antiserarecognized epitopes on the synthetic peptide 78-119. The reaction ofpeptide 78-119 with the antibodies was not unexpected because thisregion of the sequence was predicted to be exposed on the surface of theprotein and to be antigenic. Not only does this further substantiate abelief that AMP-18 or its immediate precursor, is a growth factor, forepithelial cells, but also provides a basis for analysis of thebioactive (and antigenic) regions of AMP-18, and a tool for theassessment of cell receptor number and identity. Chemical synthesis ofpeptides also makes available a convenient and rapid source ofconsiderable quantities of pure “wild-type” and “mutant” reagents forfurther cell studies. The synthetic peptide 78-119 apparently acts bythe same mechanism as the antrum protein, because their maximal effectsare not additive.

6. Sequence and Predicted Structure of the Pre-AMP Open Reading Frame

The predicted amino acid sequences for human and pig are 76% identical.The predicted signal peptides are not bold; the N-terminus of native pigAMP has been shown to be aspartate (FIG. 10).

7. Structure of the Native Protein

The ORF's of the human and pig cDNAs predicted polypeptides of similargeneral structure (FIG. 10). The predicted molecular weights for theotherwise unmodified human and pig proteins was 18.3 and 18.0respectively; these values are in good agreement with electrophoreticmobility in SDS the of antrum proteins reacting with the antisera of thepresent invention.

The antisera was used to assist in the purification of the protein fromextracts of pig antrum mucosa. Immnoaffinity methods applied to totaltissue extracts have not proven very effective, but by using immunoblotsto monitor cell-fractionation, gradient centrifugation and gelelectrophoresis sufficient amounts of the pig 18 kDa polypeptide waspurified to confirm by sequencing that the native N-terminus is onepredicted by cleavage of about 20 amino acids from the N-terminus of theORF precisely at the alanine-aspartate site anticipated for signalpeptide removal. Despite the abundance of asparagine residues, none fitthe consensus context for glycosylation. Fairly extensive regions whichmay possess amphipathic helix forming propensity. The latter mayrepresent units within the protein or as peptides after processing.Using circular dichroism the synthetic peptide representing amino acids126-143 in the human preAMP sequence (FIG. 3) is readily induced tobecome helical in moderate concentrations of trifuoroethanol conditionsused to assess helix propensity for some bioactive peptides, includinganti-microbial peptides of the magainin type (see for example Park etal., 1997).

8. Localization of AMP-18

The antisera to AMP-18 have proven to be excellent histochemical probes,reacting strongly with sections of the mouse antrum region but not withthe fundus, duodenum or intestine, confirming the results of theimmunoblots. The preimmune sera give negligible reactions even at muchhigher concentration. The AMP-18 protein appears to be concentrated inmucosal epithelial cells lining the stomach lumen, although lessersignals in cells deeper in the tissue and along the upper crypt regionssuggest that cells may begin to express the protein as they migratetoward the lumenal layer. Higher magnification of the histochemicalpreparations indicates only a general cytoplasmic staining at this levelof resolution; there are some patches of intense staining that may bethe light microscope equivalent of granule-packed regions of somelumenal surface cells seen by electron microscopy (EM). The localizationof AMP-18 in the antrum mucosa is therefore very different from thosecells synthesizing gastrin which are deep in the mucosal layer.

9. Immunoelectron Microscope Localization of the AMP-18 Antigens in theMouse Stomach Antrum Mucosal Cells

The tissue pieces were fixed in 4% formaldehyde and processed forembedding in Unicryl. Thin sections were reacted with rabbit anti-humanAMP-18 antisera (1:200); bound antibodies detected by Protein-Aconjugated to 10 nm colloidal gold. The reacted sections were stainedwith lead citrate before viewing (20,000×). The gold particles arevisible over the semi-translucent secretion granules, which appear muchmore translucent here than in the standard glutaraldehyde-osmium-eponprocedure (11,400×) because of the requirements for immuno-reactivity.Negligible background was seen on other cytoplasmic structures.

The general structure of the protein implies a possible secretory roleso a precise intracellular localization would be valuable. This requiresEM immuno-cytochemical procedures. Standard embedding and stainingmethods reveal that, as previously reported by many others, the antrumregion (e.g. Johnson and McMinn, 1970) contains mucosal epithelial cellswhich are very rich in secretory granules. Preliminary immuno-EM datashow the immune sera used at 1:200-1:800 dilution react specificallywith the secretion granules. The latter appear somewhat swollen and lesselectron opaque than in standard fixation conditions and the differencesin density are harder to discern, but overall the cell structure isquite well-preserved for stomach tissue fixed and embedded under theless stringent conditions required to preserve immuno-reactivity. At1:100 dilution, the preimmune sera exhibited negligible backgrounds withno preference for the secretion granules.

10. Growth Factor Activity on Epithelial Cell Cultures

A function for AMP-18 is that it is a growth factor at least partlyresponsible for the maintenance of a functional mucosal epithelium inthe pyloric antrum and possibly elsewhere in the stomach. Initially,stomach epithelial cell lines were not immediately available, but kidneyepithelial cell systems (Kartha et al., 1992; Aithal et al., 1994;Lieske et al., 1994) were used. A fractionated antrum mucosal cellextract was used for these experiments. Using immunoblotting as a probeto follow fractionation, on lysis of the mucosal cells scraped fromeither pig or mouse antrum, the AMP-18 antigen was recovered in the 35 Sfraction on sucrose density gradients. Such high speed supernatantfractions served as the starting material for studies on cell growth.Unexpectedly, these extracts stimulated a 50% increase in confluentrenal epithelial cells of monkey (BSC-1 cells), but had no effect onHeLa or WI-38 fibroblast cells. The stimulation of BSC-1 cells was atleast as effective as that observed with diverse polypeptide mitogens,including EGF, IGF-I, aFGF, bFGF and vasopressin, assayed at theiroptimal concentrations. Comparable growth stimulation by the antrumextracts was observed when DNA synthesis was assessed by measuring[³H]thymidine incorporation into acid-insoluble material. The biologicalactivity of the antrum extracts survived heating for 5 minutes at 65°C., and dialysis using a membrane with M_(r) cutoff of 10 kDa, whichwould eliminate most oligopeptides; this treatment removes 60-70% ofpolypeptide material, but spared AMP-18 as assayed by immunoblots. Moreimportantly, mitogenic stimulation of BSC-1 cells by the mouse or pigantrum extract was inhibited when either of two different antisera tothe human recombinant preAMP-18 (expressed in bacteria) was added to theculture medium. Preimmune sera (1:100 to 1:800) had no effect on cellgrowth, nor did they alter the mitogenic effect of the antrum extracts.These observations suggest that gastric mucosal cell AMP-18 functions asa potent mitogen for kidney epithelial cells, which do not normallyexpress this protein.

To gain further evidence that the growth-promoting activity in thepartially fractionated antrum extracts was mediated by the AMP-18protein, an aliquot of the mouse extract was subjected toSDS-polyacrylamide gel electrophoresis; the method used previously todetermine the N-terminal sequence of the natural protein. The gel wascut into 2-mm slices and each slice was extracted with 3% acetonitrilein phosphate-buffered saline containing 1% BSA. The extract supernatantswere assayed for mitogenic activity. The results indicated that oneslice containing protein in the 16-19 kDa range possessedgrowth-promoting activity. Significantly, this growth response wasblocked by the immune but not the pre-immune sera. Taken together withthe relatively low sedimentation rate of the protein, these findingsprovide additional evidence to support the conclusion that AMP-18 is anepithelial cell mitogen and that it functions as a monomer or possibly ahomotypic dimer. It also implies that the structure of the protein issuch that it can readily reacquire a native conformation after thedenaturing conditions of SDS-gel electrophoresis.

To assess the interaction of the antrum growth factor activity withother cytokines, its activity was tested to determine if it was additivewith EGF in epithelial cell cultures. EGF (50 ng/ml) added withuntreated mouse antrum extract (10 μg/ml), or heated, dialyzed pigextract (10 μg/ml) exhibited additive stimulation of mitogenesis; up to74% increase in cell number above the quiescent level; the greateststimulation observed so far for any factor using the BSC-1 cell assay.An example of this additivity is shown for an AMP-peptide and EGF on AGScells in FIG. 11. This observation suggests that AMP-18 and EGF initiateproliferation by acting on different cell surface receptors. It alsoimplies that AMP-18 growth factor activity might normally collaboratewith other autocrine and paracrine factors in the maintenance orrestitution of the epithelium. In view of the results with EGF, it islikely that AMP-18 is secreted at and acts upon the apical face (i.e.,stomach lumenal face) of the epithelial cell layer while other factors(for which EGF may serve as an example) act from the basal surface.

11. Bioactivity of Gastrokine (AMP-18) Related Peptides

The activities of synthetic peptides of the present invention areunexpected. Peptides based on the ORF of the human cDNA clone peptideswere synthesized in the University of Chicago Cancer Center Peptide CoreFacility, which checks the sequence and mass spectra of the products.The peptides were further purified by HPLC. Five relatively largeoligopeptides (of about 40 amino acids each) approximately spanning thelength of the protein without including the signal peptide, wereanalyzed. One peptide 42 amino acids long spanning amino acids lys-78 toleu-119 of the pre-AMP sequence (peptide 58-99 of the matured form ofthe protein; see Table 1), including a predicted helix andglycine-proline (GP) turns, gave good mitogenic activity. This responsewas blocked by the specific antiserum, but not by the preimmune sera.

A 14 amino acid mitogenic domain is in bold type. *Peptides areidentified by their position in the amino acid sequence of thepre-gastrokine (preAMP-18). #AA; number of amino acids in a peptide.K_(1/2); concentration for half-maximal growth stimulation. **scrambled

Overlapping inactive peptides can inhibit the activity of the mitogenicpeptides: that is, human peptides 78-88 and 87-105 block the activity ofpeptide 78-119, and while peptide 87-105 blocks the activity of peptide104-117, the peptide 78-88 does not. Peptides 78-88 and 87-105 block theactivity of the protein in stomach extracts.

12. The growth Stimulatory Domain of Gastrokine (AMP-18)

Finding that a 42-amino acid peptide representing a central region ofthe novel antrum mucosal cell protein AMP-18 had mitogenic activitysimilar in character to that of the intact protein in pig and mouseantrum extracts (Table 1), has facilitated the characterization of thebio-active region of the molecule. A peptide including amino acids atpositions 78-119, gave similar maximal stimulation of growth of theBSC-1 epithelial cell line to that given by the tissue extracts and wassimilarly inhibited by several different antisera raised in rabbits tothe bacterially-expressed complete antrum protein. The mitogenicactivity of a number of synthetic “deletion” peptides related to peptide“78-119” are summarized in Table 1. Growth activity determinations haveso far been accomplished with the kidney epithelial cell line as well asseveral gastric and intestinal lines.

The original 42 amino acid sequence of peptide 78-119 was broken intothree segments bounded by lysine (K) residues; N-terminal to C-terminalthese are peptides with amino acids at positions 78-88, 87-105 and104-117. Of these only peptide 104-117 possessed mitogenic activitygiving a similar plateau of growth stimulation but requiring a highermolar concentration than the original peptide “78-119”; this isreflected in the higher K_(1/2) value, which suggests that 14-amino acidpeptide has 30-40% of the activity of the 42-amino acid peptide. Aconclusion from this is that the smaller peptide has less bindingaffinity for a cell receptor, perhaps due to a lessened ability to formthe correct conformation, or alternatively because of the loss ofancillary binding regions. The latter notion is supported by theobservations that peptides “78-88” and “87-105” can antagonize theactivity of intact 42-mer peptide 78-119; these peptides also antagonizethe activity of antrum extracts further supporting the validity ofsynthetic peptides as a means to analyze the biological function of thenovel protein. An additional aspect of the invention is that peptide87-105, but NOT 68-88, antagonizes the activity of peptide 104-117; notethat peptide 87-105 overlaps the adjacent 104-117 sequence by tworesidues.

Taken together these results may be interpreted by a relatively simplelinear model for the growth-stimulatory region of AMP-18; viz, there isan N-terminal extended binding domain (predicted to be largely helix,the relative rigidity of which may explain the linear organization ofthe relevant sequences as determined in the cell growth studies),followed by a region high in glycine and proline with no predictedstructure beyond the likelihood of turns. It is this latter region whichcontains the trigger for growth stimulation. The specificity ofantagonism by peptides 78-88 and 87-105 may be based on whether theyoverlap or not the agonist peptides 78-119 and 104-117; for example78-88 overlaps and inhibits 78-119, but does not overlap or inhibit104-117. The specificity of competition by these peptides taken with theinactivity of the 78-119 scrambled peptide, strengthens a conclusionthat AMP-18 interacts with specific cellular components. Furtherevidence that the receptor binding region extends N-terminally frompeptide 104-117 is provided by the enhanced activity of peptide 97-117which contains a seven amino acid N-terminal extension of 104-117. Apeptide with a four amino acid extension in the C-terminal direction(peptide 104-121) appears to have slightly less activity to the parent104-117, but does include a natural tyrosine, which makes possiblelabeling with radioactive iodine, which allows determination of thebinding of AMP-related peptides to cells, initially by assessment ofnumber of binding sites and subsequently detection of the receptorprotein(s).

The peptide 97-107 was used for most tests because of its activity(equal to the 42-mer) and its relative economy (21 amino acids inlength). However, a C-terminal extension to the tyr-121 gives the mostactive peptide thus far, perhaps because it stabilizes secondarystructure. Even though this peptide does not match the nanomolaractivity of EGF, for example, it is much more potent than reported fortrefoil peptides (Podolsky, 1997). An estimate for the activity theintact AMP protein is ca. 1-10 nM.

13. Expression of Recombinant Protein

E. coli. Recombinant constructs are generally engineered bypolymerase-chain-reactions using synthetic oligonucleotidescomplementary to the appropriate regions of the full-length cDNAsequences within the PT/CEBP vector and extended by convenientrestriction enzyme sites to enable ready insertion into standard vectorpolylinkers. The initial experiments with expression of the AMP ORF inbacterial systems employed an expression vector PT/CEBP, which includedan N-terminal His6-tag (SEQ ID NO: 16) (Jeon et al., 1994), intended tofacilitate the purification of the expressed protein on Ni-NTA resin(Qiagen). Expression of the full-length human cDNA within this vector inthe host BL21(DE3)pLyS gave good yields of insoluble protein, whichafter electrophoresis under denaturing conditions was suitable for useas an immunogen in rabbits to obtain specific high-titer antibodies, butwhich has not been useful for analysis of the protein's native structureand function. This insolubility is most probably due to the presence ofan unnatural N-terminus, having a His6-tag (SEQ ID NO: 16) upstream ofhydrophobic signal peptide, in the expressed protein. Engineeringvectors which will express the ORF without the hydrophobic signalpeptide sequence are also useful. These are constructed using bacterialexpression vectors with and without N- or C-terminal His-tags. The humanAMP-18 sequence lacking the 20 amino acid signal peptide and containinga His6-tag (SEQ ID NO: 16) was also expressed in bacteria.

Pichia pastoris. Among the simple eukaryotes, the budding yeast P.pastoris is gaining wide popularity as an expression system of choicefor production and secretion of functional recombinant proteins (Romanoset al., 1992; Cregg et al., 1993). In this system, secretion of theforeign protein may utilize either its own signal peptide or the highlycompatible yeast mating-type alpha signal. This organism will correctlyprocess and secrete and at least partially modify the AMP-18 protein.Vectors for constitutive and regulated expression of foreign genes aredeveloped in Pichia (Sears et al., 1998). In addition to a poly-linkercloning site, these vectors contain either the high expressionconstitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) or themethanol-regulated alcohol oxidase promoter (AOX1). The latter is anextremely stringent promoter yielding insignificant product in normalculture conditions while giving the highest expression of the vectorstested in the presence of methanol, amounting to as much as 30% of thecell protein. The advantage that the yeast Pichia has over the mammalianand insect alternatives is that it is continuously grown in protein-freemedia, thus simplifying the purification of the expressed protein andeliminating extraneous bioactivities originating in the serum or thehost animal cells. A pIB4 construct (inducible by methanol-containingmedium) contains the complete human preAMP-18 cDNA sequence.

Baculovirus/insect cells. An alternative, frequently successful,non-mammalian eukaryotic expression system is that using recombinantBaculovirus, such as Autographa californica, in an insect cell culturesystem. As with Pichia, a large repertoire of convenient vectors areavailable in this system, containing both glutathione S-transferase(GST)-and His6-tags (SEQ ID NO: 16) (Pharmingen). Transfections arecarried out into Spodoptera frugiperda (Sƒ) cells; these cells can beslowly adapted to protein-free medium to favor the purification ofsecreted proteins. If an endogenous signal peptide does not function inthese cells, secretion of foreign proteins can also be forced usingvectors containing the viral gp67 secretion signal upstream of thecloning site. Recombinant proteins can be expressed at levels rangingfrom 0.1-50% total cell protein. Some protein modifications may be morefavored in this insect cell system relative to yeast, but still may notduplicate the mammalian system. It appears that the insect expressionsystem would be somewhat more onerous than Pichia, and not entirelysubstitute for expression in mammalian cells. The human AMP-18 sequencelacking the 20 amino acid signal peptide and containing a His6-tag (SEQID NO: 16) was expressed in Baculovirus.

Mammalian cells. Modifications not detectable by immunoblot analysis maytake place in mammalian cells that are not duplicated in cells of othereukaryotes. Although not as convenient as prokaryotic and simpleeukaryotic systems, mammalian cells are now frequently used for bothtransient and continuous expression of foreign proteins. Several growthfactors have been expressed and secreted in significant amounts usingthese systems.

The plasmid pcDNA3/human kidney 293 system: pcDNA3 contains a polylinkercloning site flanked by the strong constitutive cytomegalovirus (CMV)promoter and a SV40 polyA signal (Invitrogen). Laboratory experience isthat 60-90% transient transfection levels can be achieved. To this end,PCR amplification of the human preAMP cDNA clone is performed witholigonucleotides that contain the initiation codon and native ribosomebinding site (Kozak sequence) as well as suitable restriction enzymelinkers for correct orientation into pcDNA3. Favorable constructs wereidentified in the transient assay using the potent antibioticblasticidin S and a vector containing the resistance gene, stablemammalian transfectant cell lines can be established “in less than oneweek” (Invitrogen). The available vectors also include the constitutiveCMV promoter, a polylinker cloning site, an elective V5-epitope/His6-tag(SEQ ID NO: 16) and the SV40 poly(A) signal (PcDNA6/V5-His).

14. Expression and Analysis of Altered (Modified) Forms of AMP-18

Given an efficient expression system for the production of “wild-type”AMP-18 , a series of mutant proteins, containing either deletions orsubstitutions may be created, which permit analysis of the functionaldomains. The amphipathic helices, the conserved cystine (C) residues andthe basic amino acids doublets, which may be cleavage sites, areattractive targets. Although not as simple as an enzyme assay, themitogenesis assay is routine and replicable, and enables “mutants” to becharacterized as fast as they are constructed. Dominant negative (orpositive) “mutants” are as significant as mutations exhibiting simpleloss of function, because these imply interactions with other factorsincluding possible cell receptors.

15. Biochemical and Immunoaffinity Fractionation of Expressed and NativeGastrokine Proteins

In the case of some of the expressed forms of gastrokine AMP-18 , therecombinant protein contains peptide tags that will permit the rapidpurification of soluble protein. The presence of these tags, if they donot severely interfere with the protein's normal functions, also permitanalysis of interactions with other relevant macromolecules. His6-tags(SEQ ID NO: 16) permit purification by binding the recombinant proteinsto Ni-NTA resin beads (Janknecht et al., 1991; Ni-NTA resin fromQiagen). The tagged protein is bound with greater affinity than mostantigen-antibody complexes and can be washed rigorously before the N_(i)²⁺-histidine chelation complex is disrupted by excess imidazole torelease the purified protein. GST-tagged recombinant proteins arepurified on glutathione-agarose, washed and then eluted with reducedglutathione (Smith and Johnson, 1988). As with all the proposedexpression systems, each protein preparation may be tested at theearliest possible stage for its growth factor activity.

Conventional fractionation procedures are used to achieve the desiredpurity, particularly in the case of the isolation of the natural proteinfrom tissue. Pig antrum mucosa is a preferred starting point for thelatter, using initial centrifugation and heat-treatment protocol,followed by a size-exclusion column: BioGel P60 is suitable, given theevidence that the 18 kDa protein exists, most probably as a monomer inthe extracts. The eluant is loaded on an immunoaffinity matrix createdby crosslinking anti-AMP antibodies purified on HiTrap Protein A toCNBr-activated Sepharose 4B (Pharmacia). Further modification of theimmnoaffinity matrix may be helpful, either by extension of the linkerto the matrix, which has proven useful in the past (Aithal et al.,1994), or by crosslinking the antibody to immobilized protein-A. Becauseactive protein can be recovered by SDS-gel elution, active protein mayalso be recovered from the antigen-antibody complexes. Furtherfractionation could be achieved by C8 reversed-phase high-performanceliquid chromatography (HPLC) column. A final step is the use of theSDS-gel elution technique with confirmation of identity by N-terminalsequencing. In all of these steps the immunodetectable AMP-18 and thegrowth factor activity should fractionate together.

16. AMP-18 Related Synthetic Peptides

AMP-18 may be precursor to one or several bioactive peptides. Syntheticpeptides provide a convenient avenue to explore the function of aprotein; peptides may mimic aspects of the function or antagonize them.If a peptide either duplicates or inhibits the protein's activity, thenit suggests the identity of functional domains of the intact protein,and also provides the possibility of synthesizing specifically taggedprobes to explore protein-cell interactions.

Finding that a synthetic 42 amino acid peptide, representing a middleregion of the human protein, is capable of mimicking the growth factoractivity of the partially fractionated antrum mucosal extracts hasprovided a short-cut to the analysis of AMP-18 function. This peptide(designated peptide 58-99; amino acids are at positions 58-99 of themature protein after removal of the signal peptide) in addition toseveral possible protein processing sites at lysine pairs, contains oneof the regions capable of extended helix formation as well as aglycine-proline loop. An added advantage of this peptide is that itcontains epitopes recognized by both of the antisera disclosed herein.Some smaller peptides derived from this sequence were synthesized tofocus on the bioactive regions. Initially sequences bounded by thelysine residues were studied because they may indicate distinct domainswithin the protein structure, by virtue of being exposed on the surfaceof the protein, as witnessed by the antigenicity of this region, and maybe sites of cleavage in vivo to bioactive peptides. The glycine-prolineregion is important (see Table 1 illustrating the bioactive domains ofAMP-18). Glycine-proline sequences are known to be involved in SH3 (srchomology domain type 3) ligands (see Cohen et al., 1995; Nguyen et al.,1998); because SH domains are involved in protein-protein interactionsthat GP region of AMP-18 may be involved in the interaction of theprotein with a cell surface receptor. The exact GPGGPPP (SEQ ID NO: 24)sequence found in AMP-18 has not been reported for theintracellular-acting SH3 domains, so the intriguing possibility existsthat it represents a novel protein interaction domain for extracellularligands. A 21-mer derived from amino acids at positions 97-117 of themature sequence has activity similar to the 42-mer. This shorter peptideis useful for growth assays on various epithelial cell lines. Thispeptide does not express the epitope recognized by the antiseradisclosed herein.

All of the AMP-18 derived peptides were synthesized by the Cancer CenterPeptide Core Facility of the University of Chicago, which also confirmedthe molecular mass and amino acid sequence of the purified peptides thatare isolated by HPLC. The biological activity of peptide 78-119 not onlyprovides the basis for seeking smaller peptides with mitogenic activity,but permits amino acid substitutions that have positive or negativeeffects to be found rapidly. Inactive peptides were tested for theirability to block the function of active peptides or intact AMP-18. Thepossible inclusion of D-amino acids in the peptides (in normal orreverse order) may stabilize them to degradation while permittingretention of biological function. Further the ability to synthesizeactive peptides enables tags that facilitate studies of the nature,tissue distribution and number of cellular receptors. Such tags includeHis-6 biotin or iodinated tyrosine residues appended to the peptidesequence (several of the bioactive peptides have a naturally occurringtyrosine at the C-terminus).

Synthetic peptides also permit assessment of the role of potentialsecondary structure on function. The finding that a 4 amino acidC-terminal extension of the active peptide 97-117, predicted to promotea helix similar to that for the intact AMP-18 sequence, led to a moreactive peptide 97-121, is interesting. The helix-propensity of theseactive peptides e.g. peptide 126-143, which resembles an anti-microbialmagainin peptide, provides useful information. With respect toantimicrobial peptides, the function of the magain in class is relatedto their ability to form amphipathic helices (Boman, 1995). Syntheticpeptides that can be locked in the helical form by lactam bridges(Houston et al., 1996) enhanced biological activity; at least one pairof appropriate acidic and basic amino acid residues for lactam formationalready exist in potential helix regions of AMP-18.

Another equally significant aspect of the peptide studies is thepotential availability of specific anti-AMP-18 peptides that antagonizeits biological functions. Tissue culture studies show that sub-peptidesof the growth-promoting peptide 78-119 can antagonize the activity ofthe intact peptide (see Table 1). Peptides that can occupy cellularbinding sites but lack some essential residues for activity may blockthe action of AMP-18 and its active peptides. This makes availableanother set of reagents for the analysis of cellular receptors and forassessing receptor-ligand affinity constants. Availability of definedpeptide antagonists is useful in whole animal studies, and mayeventually serve to regulate the activity of the natural protein inhumans.

17. Interactions of AMP-18 and Related Peptides with Cells: Assessmentof Cell Growth

Non-transformed monkey kidney epithelial cell line BSC-1 and otherepithelial cell lines were used to assess effects on growth. In general,conditions were chosen for each line such that cells are grown toconfluence in plastic dishes in supplemented growth medium with minimalcalf (or fetal) serum for growth (Lieske et al., 1997); BSC-1 cellsbecome confluent at 10⁶/60 mm dish with 1% calf serum. At the start ofthe growth assay the medium on the confluent culture was aspirated andreplaced with fresh medium with minimal serum to maintain viability(0.01% for BSC-1) cells. AMP-18 preparations were added to the culturemedium and 4 days later the cell monolayer was rinsed, detached withtrypsin, and the cells were counted using a hemocytometer. Determinationof the capacity of AMP-18 to initiate DNA synthesis was measured by theincorporation of [³H]thymidine (Toback, 1980); to confirm the DNAsynthesis assay, autoradiograms of leveled cells were counted (Karthaand Toback, 1985).

The protein AMP-18 is expressed in the antrum mucosa and to a lesserextent in the adjacent corpus mucosa. However, both antrum extracts andthe active synthetic peptides stimulate proliferation of most simpleepithelial cell lines. The major criterion used, apart from cells whichmight be natural targets for AMP-18 or its peptides, was that of growthcontrol, particularly cell-density restriction. Many transformed stomachlines derived from human cancer patients are available from varioussources, but most of these do not exhibit growth control. For example, agastric AGS adenocarcinoma cell subline from Dr. Duane Smoot (HowardUniversity College of Medicine) showed a greater degree of contactinhibition, and responded well to AMP-18 and its derived peptides. Thesecells do not naturally synthesize AMP-18. Similar responses wereobserved with the non-transformed rat IEC intestinal epithelial cells(provided by Dr. Mark Musch, Dept. Medicine, University of Chicago); thelatter show excellent epithelial cell characteristics in culture(Quaroni et al., 1979; Digass et al., 1998).

18. Receptors for AMP-18 on the Surface of Epithelial Cells

Characterization of the target cell receptors of AMP-18 is intriguingbecause of the apparent existence of receptors on cells which are notexpected ever to contact this protein. Initial growth response assayswere performed on kidney-derived epithelial cell lines, which respondedwell to the stomach factor. Gastric cell lines, as well as thenon-transformed rat intestinal epithelial IEC-6 cells, were used toaddress the receptors in cells that are likely the true physiologicaltargets for the antrum factor. The specificity for the action of thisprotein in vivo likely arises from the extremely tissue specific natureof its expression, rather than that of its receptor. It is possible thatAMP-18 may interact with receptors shared with other growth factors.However, the additive growth stimulus of EGF and the antrum extractssuggest that AMP-18 may have novel receptors.

Protein molecules in cell membranes that interact with AMP-18 may besought in several different ways. Pure AMP-18 or related peptideslabeled, e.g. with biotin or radioactive iodine, are used to estimatethe number of saturatable sites on the cell surface. Scatchard analysisof the binding values as used to determine the number and affinity ofreceptors. For quantitative studies, binding is measured at increasingAMP ligand concentrations, and non-specific components are identified bymeasuring binding in the presence of excess unlabeled factor. Iodinatedgrowth factors have been cross-linked to cellular receptors enablingtheir identification (Segarini et al., 1987). Labeled AMP ligands areincubated with cells, and the bound ligand is cross-linked to thereceptors by disuccinimidyl suberate. The labeled proteins are resolvedby SDS-PAGE, and autodiography is used to visualize the cross-linkedcomplex permitting an estimate of the MW of the receptor(s). Syntheticpeptide mimics or antagonists permit studies of the cellular receptors,and their properties are reasonably inferred prior to future definitiveidentification, presumably by cloning techniques.

In addition to crosslinking studies, antibodies, or his6-tagged (SEQ IDNO: 16) AMP-18 or peptides are used to isolate cellular or mucusproteins which bind to AMP-18. As an additional approach, an immobilizedAMP-18 affinity matrix can be created by using CNMBr-activatedSepharose. As a simple beginning to the analysis of the signaltransduction pathway mediated by any cell receptor, a test to assayprotein tyrosine kinase activity in affinity isolates is available(Yarden and Ullrich, 1988; Schlessinger and Ullrich, 1992).

19. Is AMP-18 Processed to Bioactive Peptides?

The functional molecular form(s) of AMP-18 is not known. Certainly, theca. 18 kDa is the protein form which accumulates in antrum mucosalcells, and substantial amounts of polypeptides of lower MW are notdetected with the antisera, even though they do react with pepsinfragments down to ca. 10 kDa and also with the bioactive peptide 78-119(having only 42 amino acids). Having access to labeled or tagged AMP-18enables a question of whether the protein is processed in antrum mucosalextracts, or by the epithelial cells which respond to it, to beexplored.

20. Genes for AMP-18 in Man and Mouse

Using PCR techniques employing primers based on the sequence of thehuman cDNA clone, genomic clones of human and mouse preAMP-18 wereobtained. The exon/intron structure (FIG. 13) is complete. Mouse AMPexons are sufficiently similar to those of human and pig to allow asequence of the mouse gene to be assembled. Human and mouse genes havevery similar structures, the mouse gene being slightly smaller. The ORFcontained in exons of the mouse gene predicts a protein having 65%identity to the human and pig proteins. A 2 kb of sequence is upstreamof the human gene.

21. Knockout of the AMP-18 Gene in Mouse

From the mouse map a targeting construct is designed. The constructpreferably contains: [5′-TK (a functional thymidine kinase gene)—ca. 5kb of the 5′ end of AMP-18 DNA—the neomycin phosph-transferase (neo)gene under the control of the phosphoglycerate kinase (PGK) promoter—ca.3 kb of the 3′ end of the gene-3′]. A considerable length of homology ofthe construct with the resident AMP-18 gene is required for efficienttargeting. Increasing the total homology from 1.7 to 6.8 kb increasesthe efficiency of homologous targeting into the hrpt gene about 200-fold(Hasty et al., 1991). Beyond that total length, the efficiency increasesonly slightly. To facilitate the detection of homologous intergrants bya PCR reaction, it is useful to have the neo gene close to one end ofthe vector. The resulting transfectants can be provided by PCR with twoprimers, one in the neo gene and the other in the AMP-18 locus justoutside of the targeting vector. Flanks extending 4 kb 5′ and 4.5 kb 3′of the mouse gene have been obtained. Through homologous recombination,the coding region will be replaced by the neo gene to ensure a completeknockout of the gene are already cloned. After trimming off the plasmidsequence, the targeting cassette is transfected into ES cells and stabletransfectants obtained by selection with G418, an analog of neomycin,and gancyclovir (Mansour et al., 1988). Southern blots with the probefrom the flanking sequence will be used to screen for targetedhomologous recombinants. Correctly targeted ES cell clones will beinjected in blastocysts from C57BL/6 mice.

Male offspring obtained from surrogate mothers that have at least 50%agouti coat (embryonic stem cell (ES) cell derived) are bred withC57BL/6 mice. F1 mice that are agouti have the paternal componentderived from the ES cells (agouti is dominant over black). 50% of thesemice should have the knockout preAMP-18 allele. These hemizygous miceare monitored for any effect of diminished gene dosage. Homozygousknockouts are preferable. If the sole function of AMP-18 is in thestomach following birth, then viable homozygotes are expected. If thesecannot be obtained, a fetally lethal defect would be indicated, and thefetal stage of abortion would be ascertained. This result would suggestan unanticipated role of the protein in normal development.

Homozygous AMP-18 knockout mice are useful for investigations of stomachmorphology and function. It is expected that such knockouts will show ifAMP-18 is essential, and at which stage of gastro-intestinal developmentit is bioactive. It is possible that the AMP-18 knockout hemizygous micewill already show a phenotype. This could occur if reduced dosage of theprotein reduces or eliminates its function, or if parental imprinting orrandom mono-allelic expression has a significant influence. A range ofpossible outcomes of the AMP-18 knockout in mice include: i) no viablehomozygotes, implying an essential unanticipated developmental role; ii)viable homozygotes, but with obviously impaired gastrointestinalfunctions; iii) no strong phenotype, i.e. the protein is not importantto the development and life of the laboratory mouse. If appropriate, thegeneration of AMP-18 in overexpressing mice is pursued. A truncatedAMP-18 protein produced in the mice could potentially create a dominantnegative phenotype; knowledge gained from the experiments will furtherdefine the functional domains of the protein. TABLE 1 BIOACTIVITY OFSYNTHETIC PEPTIDES BASED ON THE SEQUENCE OF PRE-GASTROKINE (PRE-AMP-18)Name of Peptide Sequence in #AA AMINO ACID SEQUENCE K_(1/2), μM Human 78-119 42 KKTCIVHKMKKEVMPSIQSLDALVKEKKLQGKGPGGPPPKGL 0.3 (SEQ ID NO:17)  78-88 11 KKTCIVHKMKK Inactive (SEQ ID NO:14)  87-105 19KKEVMPSIQSLDALVKEKK Inactive (SEQ ID NO: 15) 104-117 14 KKLQGKGPGGPPPK0.8 (SEQ ID NO: 11) 104-111 18 KKLQGKGPGGPPPKGLMY 1.0 (SEQ ID NO: 18) 97-117 21 LDALVKEKKLQGKGPGGPPPK 0.3 (SEQ ID NO: 12)  97-117** 21GKPLGQPGKVPKLDGKEPLAK Inactive (SEQ ID NO: 19)  97-121 25LDALVKEKKLQGKGPGGPPPKGLMY 0.2 (SEQ ID NO: 13) 109-117  9 KGPGGPPPK 2.5(SEQ ID NO: 20) 104-109  6 KKLQGK 7.4 (SEQ ID NO: 21) 110-113  4 GPGGInactive (SEQ ID NO: 22) mouse  97-119 23 LDTMVKEQK . . . GKGPGGAPPKDLMY0.2 (SEQ ID NO: 23)

TABLE 2 ABBREVIATIONS FOR AMINO ACIDS Three-letter One-letter Amino acidabbreviation symbol Alanine Ala A Arginine Arg R Asparagine Asn NAspartic acid Asp D Asparagine or aspartic acid Asx B Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glutamine or glutamic acid Glx ZGlycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine LysK Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Documents Cited

The following documents are incorporated by reference to the extent theyrelate to or describe materials or methods disclosed herein.

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1. An inhibitor of a protein, said protein selected from a group ofisolated homologous cellular growth stimulating proteins designatedgastrokines, said protein produced by gastric epithelial cells andcomprising a consensus amino acid sequence selected from the groupconsisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10),VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK(SEQ ID NO: 26), said inhibitor selected from the group of peptideshaving an amino acid sequence consisting of KKTCIVHKMKK (SEQ ID NO: 14)and KKEVMPSIQSLDALVKEKK (SEQ ID NO: 15).
 2. A pharmaceutical compositionused for the treatment of a gastro-intestinal disorder, said compositioncomprising a growth stimulating peptide derived from a gastrokineprotein.
 3. The pharmaceutical composition of claim 2 wherein thegastrokine protein comprises a consensus amino acid sequence selectedfrom the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK, (SEQ ID NO: 10)VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK.(SEQ ID NO: 26)


4. The pharmaceutical composition of claim 2 comprising a growthstimulating peptide selected from the group consisting of amino acidsequences KKLQGKGPGGPPPK, (SEQ ID NO: 11) LDALVEKKLQGKGPGGPPPK, (SEQ IDNO: 12) and LDALVEKKLQGKGPGGPPPKGLMY. (SEQ ID NO: 13)


5. A pharmaceutical composition for the treatment of diseases associatedwith overgrowth of gastric epithelia, said compositions comprising aninhibitor according to claim
 1. 6. The pharmaceutical composition ofclaim 5, wherein diseases are diseases of the colon and small intestine,said diseases selected from the group consisting of ulcerative colitisand Crohn's Disease.
 7. An isolated cDNA molecule encoding a humanprotein, said protein having the amino acid sequence as shown in FIG. 2.8. An isolated DNA molecule comprising the genomic sequence found in DNAderived from a mouse, said nucleotide sequence shown in FIG.
 4. 9. Amouse with a targeted deletion in a nucleotide sequence in the mousegenome that when expressed without the deletion encodes a protein of thegroup of gastrokine proteins comprising a consensus amino acid sequenceselected from the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ IDNO: 10), VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) andVKE(K/Q)KGKGPGG(P/A)PPK (SEQ ID NO: 26).
 10. A method of making apeptide or protein derived from a gastrokine protein, the gastrokineprotein comprising a consensus amino acid sequence selected from thegroup consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10),VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25)and VKE(K/Q)KGKGPGG(P/A)PPK(SEQ ID NO: 26), said method comprising: (a) obtaining an isolated cDNAmolecule comprising a sequence encoding the protein or peptide; (b)placing the molecule in a recombinant DNA expression vector; (c)transecting a host cell with the recombinant DNA expression vector (d)providing environmental conditions allowing the transfected host cell toproduce a protein encoded by the cDNA molecule; and (e) purifying theprotein from the host cell.
 11. A method to inhibit cellular growthstimulating activity of a protein derived from a gastrokine proteincomprising a consensus amino acid sequence selected from the groupconsisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10),VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK(SEQ ID NO: 26), said method comprising: (f) contacting the protein withan inhibitor; and (g) providing environmental conditions suitable forcellular growth stimulating activity of the protein.
 12. The method ofclaim 11, wherein the inhibitor is an antibody directed toward at leastone epitope of the protein, said epitope comprising an amino acidsequence from position 78 to position 119 of the deduced amino acidsequence in FIG.
 3. 13. The method of claim 11, wherein the inhibitor isselected from the group of inhibitor peptides consisting of KKTCIVHKMKK(SEQ ID NO: 14) and KKEVMPSIQSLDALVKEKK (SEQ ID NO: 15).
 14. A method oftesting the effects of different levels of expression of a gastrokineprotein, on mamrnalian gastrointestinal tract epithelia, said methodcomprising: (a) obtaining a mouse in accord with claim 9; (b)determining the effects of a lack of the protein in the mouse; (c)administering increasing levels of the protein to the mouse; and (d)correlating changes in the gastrointestinal tract epithelia with thelevels of the protein in the epithelia.
 15. A method to stimulatemigration of epithelial cells after injury to the gastrointestinal tractof mammals, said method comprising: (a) contacting the epithelial cellswith a composition comprising a protein derived from a gastrokineprotein, or a peptide derived from the protein; and (b) providingenvironmental conditions allowing migration of the epithelial cells. 16.A method for cytoprotection of damaged epithelial cells in thegastrointestinal tract of mammals, said method comprising: (h)contacting the damaged epithelial cells with a composition comprising agastrokine protein comprising a consensus amino acid sequence selectedfrom the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10),VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK(SEQ ID NO: 26); and (i) providing environmental conditions allowingrepair of the epithelial cells.
 17. The method of claim 16, wherein thedamaged cells are an ulcer.
 18. A method to prevent or decrease thefrequency or intensity of acute attacks of inflammatory bowel disease(IBD) in a subject in need thereof, the method comprising: (a) obtainingAMP peptide 77-97 (SEQ ID NO:12) or AMP-18 protein; and (b)administering the peptide or protein to the subject.
 19. A method tospeed recovery of barrier function and structure needed to repair theinjured colonic mucosa in subjects with established inflammatory boweldisease (IBD), the method comprising: (a) obtaining peptide AMP 77-97(SEQ ID NO:12) or AMP-18 protein; and (b) administering the peptide orprotein to the subject.
 20. The method of claim 19 further comprisingsubjects that are infants with necrotizing enterocolitis.
 21. A methodto prevent or reduce in a subject in need thereof recurrences of gastricand duodenal infections, inflammation and ulceration caused by H.pylori, the method comprising: (a) obtaining peptide AMP77-97 (SEQ IDNO:12) or AMP-18 protein; and (b) administering the peptide or proteinto subject in need thereof.
 22. A method to alleviate cancer-therapyinduced mucositis, the method comprising: (a) obtaining peptide AMP77-97(SEQ ID NO:12) or AMP-18 protein; and (b) administering the peptide orprotein to a subject in need thereof.
 23. A method to repair injuries tothe gastric mucosa comprising: (a) obtaining peptide AMP77-97 (SEQ IDNO:12) or AMP-18 protein; and (b) administering the peptide or proteinto a subject in need thereof.
 24. The method of claim 23 where theinjuries are gastritis or gastric ulcers.
 25. The method of claim 23wherein the injuries are caused by non-steroidal anti-inflammatory drugsor alcohol.
 26. A method to speed recovery of a subject from acute renalfailure the method comprising; (a) obtaining peptide AMP77-97 (SEQ IDNO:12) or AMP-18 protein; and (b) administering the peptide or proteinto the subject in need thereof.